Endoribonuclease compositions and methods of use thereof

ABSTRACT

The present disclosure provides variant Csy4 endoribonucleases, nucleic acids encoding the variant Csy4 endoribonucleases, and host cells genetically modified with the nucleic acids. The variant Csy4 endoribonucleases find use in a variety of applications, which are also provided. The present disclosure also provides methods of detecting a specific sequence in a target polyribonucleotide; and methods of regulating production of a target RNA in a eukaryotic cell.

CROSS-REFERENCE

This application is a divisional of U.S. patent application Ser. No.13/671,120, and claims the benefit of International Application No.PCT/US2011/035775, filed May 9, 2011, U.S. Provisional PatentApplication No. 61/333,163, filed May 10, 2010, U.S. Provisional PatentApplication No. 61/365,627, filed Jul. 19, 2010, and U.S. ProvisionalPatent Application No. 61/413,287, filed Nov. 12, 2010, each of whichapplications is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. T32GM07232 awarded by the National Institutes of Health and Grant No.MCB-0950971 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“BERK-118CIP_Sequence_Listing” created on Jun. 9, 2015 and having a sizeof 124 KB. The contents of the text file are incorporated by referenceherein in their entirety.

BACKGROUND

DNA restriction enzymes transformed molecular biology in the 1970s bymaking it possible to cleave specific DNA sequences at will. Sequencingof RNA molecules currently entails copying the RNA into a DNA strandthat is then sequenced by conventional methods. This approach, alsoknown as RNASeq, is robust and can yield many millions of sequencereads. However, the necessity of generating cDNA introduces inherentbias due to sequence-dependent efficiencies of individual steps.

LITERATURE

-   Carte et al. (2008) Genes Dev. 22:3489; U.S. Patent Publication No.    2010/0093026.

SUMMARY OF THE INVENTION

The present disclosure provides variant Csy4 endoribonucleases, nucleicacids encoding the variant Csy4 endoribonucleases, and host cellsgenetically modified with the nucleic acids. The variant Csy4endoribonucleases find use in a variety of applications, which are alsoprovided. The present disclosure also provides methods of detecting aspecific sequence in a target polyribonucleotide; and methods ofregulating production of a target RNA in a eukaryotic cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict specific recognition of a pre-crRNA substrate byPa14Csy4. The nucleotide sequence depicted is5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′ (SEQ ID NO:1).

FIGS. 2A-2C depict crystal structures of Csy4 bound to RNA substrate.

FIGS. 3A and 3B depict: a detailed view of the catalytic center of Csy4(FIG. 3A); and cleavage activity of Csy4 wild-type (WT) and mutants(FIG. 3B).

FIG. 4 depicts invariant amino acids among 12 Csy4 sequences. Pa (SEQ IDNO:8); Yp (SEQ ID NO:34); Ec89 (SEQ ID NO:39); Dn (SEQ ID NO:79); Ab(SEQ ID NO:84); MP1 (SEQ ID NO:2); MP01 (SEQ ID NO:3); SW (SEQ ID NO:4);Pm (SEQ ID NO:85); Pw (SEQ ID NO:13); and Dd (SEQ ID NO:10).

FIGS. 5A-5BD present an amino acid sequence alignment of various Csy4polypeptides, as well as the nucleotide sequences of RNA sequencesrecognized by each Csy4 polypeptide.

FIG. 6 depicts examples of amino acid sequences of enzymaticallyinactive, sequence-specific endoribonucleases.

FIG. 7 depicts an example of a method for detecting a specific sequencein a target polyribonucleotide.

FIG. 8 depicts the effect of imidazole on activation of variousenzymatically inactive Csy4 variants.

FIG. 9 depicts an exemplary method of isolating a target RNA. A Csy4target stem-loop (SEQ ID NO:103) is shown.

FIG. 10 depicts an exemplary method of regulating expression of a targetRNA in a eukaryotic cell. A Csy4 RNA substrate sequence (SEQ ID NO:103)is shown.

FIG. 11A-11G depicts sequence-specific Csy4 endoribonucleases andmutant, inactive versions of these endoribonucleases that can bereactivated in the presence of imidazole. The mutated residue isinferred from the conserved histidine (starred) of the alignmentdepicted in FIG. 4. This figure also depicts the cognate RNA substratesfor the Csy4 variants.

FIGS. 12A-12E illustrate that imidazole can restore cleavage activity toa variety of Csy4 enzymes comprising histidine to alanine active sitemutations.

FIGS. 13A-13B depict cleavage rate constants (before and afternormalization) for six Csy4 variants paired with nine possible RNAsubstrates.

DEFINITIONS

As used herein, “polyribonucleotide” refers to a polymeric form ofribonucleotides, and includes RNA, RNA containingdeoxyribonucleotide(s), and DNA containing ribonucleotide(s). Apolyribonucleotide can in some cases include one or more modifiednucleotides (e.g., deoxyinosine, deoxyuridine orhydroxymethyldeoxyuridine). In some cases, a polyribonucleotide consistsof a ribonucleotides only (i.e., does not include anydeoxyribonucleotides). In some cases, a polyribonucleotide comprisesribonucleotides, and one or more modified ribonucleotides, but does notinclude any deoxyribonucleotides. In other cases, a polyribonucleotidecomprises ribonucleotides, and may comprise one or more modifiedribonucleotides, and one or more deoxyribonucleotides (includingmodified deoxyribonucleotides). In some cases, where apolyribonucleotide comprises one or more deoxyribonucleotides, thedeoxyribonucleotides comprise from about 50% to about 40%, from about40% to about 30%, from about 30% to about 20%, from about 20% to about10%, from about 10% to about 1%, or less than 1%, of the totalnucleotides in the polyribonucleotide.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyand refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides,vectors, probes, and primers.

A “biological sample” encompasses a variety of sample types obtainedfrom a cell, extracellular matter, a tissue, or a multicellularorganism. The definition encompasses blood and other liquid samples ofbiological origin, solid tissue samples such as a biopsy specimen ortissue cultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid (e.g., cerebrospinal fluid,bronchoalveolar lavage fluid, urine, blood, a blood fraction (e.g.,plasma; serum), sputum, and the like), and tissue samples. In somecases, a biological sample comprises cells. In other cases, a biologicalsample is cell free.

The term “operably linked” refers to functional linkage betweenmolecules to provide a desired function. For example, “operably linked”in the context of nucleic acids refers to a functional linkage betweennucleic acids to provide a desired function such as transcription,translation, and the like, e.g., a functional linkage between a nucleicacid expression control sequence (such as a promoter, signal sequence,or array of transcription factor binding sites) and a secondpolynucleotide, wherein the expression control sequence affectstranscription and/or translation of the second polynucleotide. “Operablylinked” in the context of a polypeptide refers to a functional linkagebetween amino acid sequences (e.g., of different domains) to provide fora described activity of the polypeptide.

“Isolated” refers to a protein or nucleic acid that, if naturallyoccurring, is in an environment different from that in which it maynaturally occur. “Isolated” is meant to include proteins or nucleicacids that are within samples that are substantially enriched for theprotein or nucleic acid of interest and/or in which the protein ornucleic acid of interest is partially or substantially purified. Wherethe protein or nucleic acid is not naturally occurring, “isolated”indicates the protein or nucleic acid has been separated from anenvironment in which it was made by either synthetic or recombinantmeans.

“Substantially pure” indicates that an entity (e.g., polypeptide or anucleic acid) makes up greater than about 50% of the total content ofthe composition (e.g., total protein of the composition) and typically,greater than about 60% of the total protein content. In someembodiments, “substantially pure” refers to compositions in which atleast 75%, at least 85%, at least 90% or more of the total compositionis the entity of interest (e.g. 95%, of the total protein). In someembodiments, the protein or nucleic acid of interest will make upgreater than about 90%, greater than about 95%, greater than about 98%,or greater than about 99%, of the total protein or nucleic acid in thecomposition.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asite-specific endoribonuclease” includes a plurality of suchsite-specific endoribonucleases and reference to “the targetpolyribonucleotide” includes reference to one or more targetpolyribonucleotides and equivalents thereof known to those skilled inthe art, and so forth. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides variant Csy4 endoribonucleases, nucleicacids encoding the variant Csy4 endoribonucleases, and host cellsgenetically modified with the nucleic acids. The variant Csy4endoribonucleases find use in a variety of applications, which are alsoprovided. The present disclosure also provides methods of detecting aspecific sequence in a target polyribonucleotide; and methods ofregulating production of a target RNA in a eukaryotic cell.

Methods of Detecting a Sequence in a Target Polyribonucleotide

The present disclosure provides a method of detecting a sequence in atarget polyribonucleotide. The methods are useful for detecting thepresence of a particular sequence in a polyribonucleotide, and cantherefore be used to detect a polyribonucleotide comprising a particularsequence. For example, the method can be used to detect the presence ofa polyribonucleotide of a pathogen in a sample (e.g., in a biologicalsample).

A subject method can detect as few as 100 copies, down to a single copy,of a target polyribonucleotide. Thus, e.g., a subject method can detectfrom 1 to about 5, from about 5 to about 10, from about 10 to about 50,or from about 50 to about 100, or more than 100, copies of a targetpolyribonucleotide in a sample (e.g., in a single cell, in a singleembryo, or other biological sample). A subject method is thus useful forvarious forensic, research, and diagnostic applications.

In some embodiments, a subject method of detecting a specific sequencein a target polyribonucleotide comprises: a) contacting the targetpolyribonucleotide with a oligonucleotide probe comprising the specificsequence and an enzymatically active sequence-specific Csy4endoribonuclease under conditions that favor duplex formation betweenthe oligonucleotide probe and the target polyribonucleotide, wherein theduplex is cleaved by the Csy4 endoribonuclease; and b) detectingspecific binding between the oligonucleotide probe and the targetpolyribonucleotide, wherein detection of duplex formation between theoligonucleotide probe and the target polyribonucleotide indicates thepresence of the specific sequence in the target polyribonucleotide.

In some cases, the oligonucleotide probe is linked to a peptide, and thepeptide is released upon cleavage of the duplex by the Csy4endoribonuclease; in these cases, the detection step involves detectionof the released peptide. For example, the released peptide is detectedby binding to an antibody specific for the peptide, e.g., where theantibody is immobilized. In some embodiments, the targetpolyribonucleotide is immobilized on a solid support. Targetpolyribonucleotides include any of a variety of polynucleotides, e.g.,the target polyribonucleotide can be a polyribonucleotide of a pathogen.

As noted above, in some embodiments, the antibody or the targetpolynucleotide is immobilized on a solid support (insoluble support).Suitable insoluble supports include, but are not limited to agarosebeads, magnetic beads, a test strip, a multi-well dish, and the like.The insoluble support can comprise a variety of substances (glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amylose, natural and modified celluloses,polyacrylamides, agaroses, and magnetite) and can be provided in avariety of forms, including, e.g., agarose beads, polystyrene beads,latex beads, magnetic beads, colloid metal particles, glass and/orsilicon chips and surfaces, nitrocellulose strips, nylon membranes,sheets, wells of reaction trays (e.g., multi-well plates), plastictubes, etc.

In some embodiments, the method generally involves: a) contacting atarget polyribonucleotide with a sequence-specific endoribonuclease; andb) detecting cleavage fragments produced by site-specific cleavage ofthe target polyribonucleotide, where production of cleavage fragmentsexpected upon cleavage at a specific sequence in the polyribonucleotideindicates the presence of the specific sequence.

In other embodiments, a subject method of detecting a sequence in atarget polyribonucleotide involves: a) contacting a targetpolyribonucleotide with: i) a sequence-specific endoribonuclease; andii) an oligonucleotide probe comprising a linked detection moiety, wherethe oligonucleotide probe comprises a specific, known nucleotidesequence; wherein the oligonucleotide probe forms a duplex with acomplementary sequence in the target polyribonucleotide based on bindingof the known nucleotide sequence present in the oligonucleotide probe toa complementary sequence in the target polyribonucleotide, and where thesequence-specific endoribonuclease cleaves the duplex in asequence-specific manner, thereby releasing the detection moiety fromthe oligonucleotide probe; and b) detecting the released detectionmoiety, where release of the detection moiety indicates the presence ofthe specific sequence. In some embodiments, two or more differentoligonucleotide probes are used, each comprising a different specific,known nucleotide sequence.

In some embodiments, the detection moiety is a polypeptide. Thepolypeptide can be detected using an immunological assay (e.g., anenzyme-linked immunosorbent assay (ELISA); a radioimmunoassay (RIA);etc.), using an antibody specific for the polypeptide detection moiety.The antibody specific for the polypeptide detection moiety can comprisea detectable label. The immunological assay can be carried out on a teststrip (e.g., in a lateral flow assay) or other suitable medium such as amulti-well plate.

In some embodiments, the detection moiety is a fluorescent protein,where suitable fluorescent proteins are as described herein. In otherembodiments, the detection moiety is luciferin or other substrate forluciferase. Suitable luciferins or other luciferase substrates include,e.g., luciferin (e.g., a firefly luciferin); an aminoluciferin;coelenterazine; a modified coelenterazine as described in U.S. Pat. No.7,537,912; a coelenterazine analog as described in U.S. PatentPublication No. 2009/0081129 (e.g., a membrane permeant coelenterazineanalog as described in U.S. Patent Publication No. 2009/0081129, e.g.,one of Structures II, III, IV, V, and VI of U.S. Patent Publication No.2009/0081129); aminoluciferin; dihydroluciferin; luciferin 6′methylether; or luciferin 6′ chloroethylether. See, e.g., Branchini, B.R. et al. Anal. Biochem. 2010, 396, 290-296; and Mezzanotte, L. et al.,In vivo bioluminescence imaging of murine xenograft cancer models with ared-shifted thermostable luciferase. Mol. Imaging Biol. (2009, Nov. 9,online; PubMed ID: 19937390).

A non-limiting example of a subject detection method is illustratedschematically in FIG. 7. In the example depicted in FIG. 7, smalloligonucleotides that bind discrete regions of a target polynucleotide(e.g., a viral RNA) are contacted with the target polynucleotide, wherethe oligonucleotides comprise detectable moieties (e.g., ligands;peptides; etc.). An enzymatically active, sequence-specific restrictionendonuclease (RRE) that targets the oligonucleotide/viral RNA duplex isadded. The enzyme cleaves the oligonucleotide/viral RNA duplex; andligands are released for detection. The enzyme cleaves further duplexes,thereby amplifying the signal. Released ligands are detected using alateral flow (e.g., test strip) or an immunological based assay (e.g.,ELISA).

A suitable sequence-specific endoribonuclease is an enzymaticallyactive, sequence-specific endoribonuclease. Endoribonucleases that aresuitable for use in a subject detection method include endoribonucleasesthat bind to and cleave a substrate polyribonucleotide in asequence-specific manner include enzymatically active polypeptides andthat have at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence set forth in FIG. 4 (Csy4 amino acidsequences).

Endoribonucleases that are suitable for use in a subject detectionmethod include endoribonucleases that bind to and cleave a substratepolyribonucleotide in a sequence-specific manner include enzymaticallyactive polypeptides and that have at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence set forthin FIG. 5 or FIG. 11 (SEQ ID NO: 39, 79, 84, 90, 104, 108, or 110) (Csy4amino acid sequences). FIG. 5 provides sequences specifically bound bythe various endoribonucleases. In some cases, a suitable enzymaticallyactive sequence-specific Csy4 endoribonuclease can comprise an aminoacid sequence of a Csy4 amino acid sequence depicted in FIG. 5 or FIG.11 (SEQ ID NO: 39, 79, 84, 90, 104, 108, or 110).

Endoribonucleases that are suitable for use in a subject detectionmethod include endoribonucleases that bind to and cleave a substratepolyribonucleotide in a sequence-specific manner include enzymaticallyactive polypeptides and that differ from an amino acid sequence setforth in any one of FIGS. 4, 5, and/or 11 by from 1 to 20 (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)amino acid substitutions and/or insertions and/or deletions.

The target polyribonucleotide to be detected can be present in a sample,e.g., a biological sample such as blood, a blood product (e.g., plasma),urine, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, atissue, cells, etc. The target polyribonucleotide can be isolated orpurified. The target polyribonucleotide can be a messenger RNA (mRNA), aviral RNA, bacterial RNA, parasite RNA, or other RNA species. Viral RNAsinclude, but are not limited to, any member of the Flaviviridae, e.g.,hepatitis C virus, Dengue virus, Yellow Fever Virus, West Nile Virus,etc.; any member of Retroviridae; an immunodeficiency virus (e.g., humanimmunodeficiency virus); etc.

The target polyribonucleotide to be detected can be present in a cell ofa multicellular organism (or can be obtained from a cell of amulticellular organism).

The target polyribonucleotide to be detected can be present in orobtained from a cell or organism of any of the six kingdoms, e.g.,Bacteria (e.g., Eubacteria); Archaebacteria; Protista; Fungi; Plantae;and Animalia. Suitable sources of target polyribonucleotides includeplant-like members of the kingdom Protista, including, but not limitedto, algae (e.g., green algae, red algae, glaucophytes, cyanobacteria);fungus-like members of Protista, e.g., slime molds, water molds, etc.;animal-like members of Protista, e.g., flagellates (e.g., Euglena),amoeboids (e.g., amoeba), sporozoans (e.g, Apicomplexa, Myxozoa,Microsporidia), and ciliates (e.g., Paramecium). Suitable sources oftarget polyribonucleotides include members of the kingdom Fungi,including, but not limited to, members of any of the phyla:Basidiomycota (club fungi; e.g., members of Agaricus, Amanita, Boletus,Cantherellus, etc.); Ascomycota (sac fungi, including, e.g.,Saccharomyces); Mycophycophyta (lichens); Zygomycota (conjugationfungi); and Deuteromycota. Suitable sources of targetpolyribonucleotides include members of the kingdom Plantae, including,but not limited to, members of any of the following divisions: Bryophyta(e.g., mosses), Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g.,liverworts), Lycophyta (e.g., club mosses), Sphenophyta (e.g.,horsetails), Psilophyta (e.g., whisk ferns), Ophioglossophyta,Pterophyta (e.g., ferns), Cycadophyta, Gingkophyta, Pinophyta,Gnetophyta, and Magnoliophyta (e.g., flowering plants). Suitable sourcesof target polyribonucleotides include members of the kingdom Animalia,including, but not limited to, members of any of the following phyla:Porifera (sponges); Placozoa; Orthonectida (parasites of marineinvertebrates); Rhombozoa; Cnidaria (corals, anemones, jellyfish, seapens, sea pansies, sea wasps); Ctenophora (comb jellies);Platyhelminthes (flatworms); Nemertina (ribbon worms); Ngathostomulida(jawed worms); Gastrotricha; Rotifera; Priapulida; Kinorhyncha;Loricifera; Acanthocephala; Entoprocta; Nemotoda; Nematomorpha;Cycliophora; Mollusca (mollusks); Sipuncula (peanut worms); Annelida(segmented worms); Tardigrada (water bears); Onychophora (velvet worms);Arthropoda (including the subphyla: Chelicerata, Myriapoda, Hexapoda,and Crustacea, where the Chelicerata include, e.g., arachnids,Merostomata, and Pycnogonida, where the Myriapoda include, e.g.,Chilopoda (centipedes), Diplopoda (millipedes), Paropoda, and Symphyla,where the Hexapoda include insects, and where the Crustacea includeshrimp, hill, barnacles, etc.; Phoronida; Ectoprocta (moss animals);Brachiopoda; Echinodermata (e.g. starfish, sea daisies, feather stars,sea urchins, sea cucumbers, brittle stars, brittle baskets, etc.);Chaetognatha (arrow worms); Hemichordata (acorn worms); and Chordata.Suitable members of Chordata include any member of the followingsubphyla: Urochordata (sea squirts; including Ascidiacea, Thaliacea, andLarvacea); Cephalochordata (lancelets); Myxini (hagfish); andVertebrata, where members of Vertebrata include, e.g., members ofPetromyzontida (lampreys), Chondrichthyces (cartilaginous fish),Actinopterygii (ray-finned fish), Actinista (coelocanths), Dipnoi(lungfish), Reptilia (reptiles, e.g., snakes, alligators, crocodiles,lizards, etc.), Aves (birds); and Mammalian (mammals). Suitable plantsinclude any monocotyledon and any dicotyledon.

Thus, e.g., a target polyribonucleotide can be present in or obtainedfrom cells from organisms that include, but are not limited to, aprotozoan, a plant, a fungus, an algal cell, a yeast, a reptile, anamphibian, a mammal, a marine microorganism, a marine invertebrate, anarthropod, an isopod, an insect, an arachnid, an archaebacterium, and aeubacterium.

A target polyribonucleotide can be present in or obtained from anon-human embryo, e.g., a Drosophila embryo; a zebrafish embryo; a mouseembryo; etc.

A target polyribonucleotide can be present in or obtained from a stemcell, e.g., an in vitro stem cell; a non-human stem cell; etc. Suitablestem cells include embryonic stem cells, adult stem cells, and inducedpluripotent stem (iPS) cells.

In some embodiments, target polyribonucleotide will be isolated from atissue taken from an organism; from a particular cell or group of cellsisolated from an organism; etc. For example, where the organism is aplant, the target polyribonucleotide will in some embodiments beisolated from the xylem, the phloem, the cambium layer, leaves, roots,etc. Where the organism is an animal, the target polyribonucleotide willin some embodiments be isolated from a particular tissue (e.g., lung,liver, heart, kidney, brain, spleen, skin, fetal tissue, etc.), or aparticular cell type (e.g., neuronal cells, epithelial cells,endothelial cells, astrocytes, macrophages, glial cells, islet cells, Tlymphocytes, B lymphocytes, etc.).

Methods of Regulating Production of a Target RNA

The present disclosure provides a method of regulating production of atarget RNA in a cell. The method generally involves contacting agenetically modified host cell with an agent that activates an induciblepromoter, where the genetically modified host cell is geneticallymodified with a recombinant expression vector comprising a nucleotidesequence encoding an enzyme that catalyzes cleavage at asequence-specific cleavage site in a substrate polyribonucleotide, wherethe enzyme-encoding nucleotide sequence is operably linked to theinducible promoter, and where, upon activation of the induciblepromoter, the enzyme is produced in the cell and cleaves said target RNAfrom a precursor RNA.

FIG. 10 provides a schematic depiction of an exemplary method ofregulating production of a target RNA. In FIG. 10, an endogenous targetRNA is modified to include a Csy4 RNA substrate (e.g.,GUUCACUGCCGUAUAGGCAG (SEQ ID NO:103); or SEQ ID NO:1) in the 3′untranslated region (3′ UTR). Cys4 expression in the host cell leads tobinding and cleavage of the RNA substrate. The cleaved RNA now lacks itspolyA tail and will be degraded.

For example, in some embodiments, the present disclosure provides amethod of regulating production of a target RNA in a eukaryotic cell,where the method involves contacting a genetically modified host cellwith an agent that activates an inducible promoter, where thegenetically modified host cell is genetically modified with arecombinant expression vector comprising a nucleotide sequence encodingan enzymatically active sequence-specific Csy4 endoribonuclease thatcatalyzes cleavage at a sequence-specific cleavage site in a substratepolyribonucleotide, where the enzyme-encoding nucleotide sequence isoperably linked to the inducible promoter, and where, upon activation ofthe inducible promoter, the enzyme is produced in the cell and cleavessaid target RNA from a precursor RNA. In some cases, the target RNAspecies is a regulatory RNA. In some cases, cleavage of said target RNAfrom a precursor RNA inactivates the precursor RNA.

A suitable sequence-specific endoribonuclease is an enzymaticallyactive, sequence-specific endoribonuclease. Endoribonucleases that aresuitable for use in a subject method of regulating production of atarget RNA include endoribonucleases that bind to and cleave a substratepolyribonucleotide in a sequence-specific manner include enzymaticallyactive polypeptides and that have at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to an amino acid sequence set forthin FIG. 4 (Csy4 amino acid sequences).

Endoribonucleases that are suitable for use in a subject method ofregulating production of a target RNA include endoribonucleases thatbind to and cleave a substrate polyribonucleotide in a sequence-specificmanner include enzymatically active polypeptides and that have at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to an aminoacid sequence set forth in FIG. 5 or FIG. 11 (SEQ ID NO: 39, 79, 84, 90,104, 108, or 110) (Csy4 amino acid sequences). FIG. 5 and FIG. 11provide sequences specifically bound by the various endoribonucleases.

Endoribonucleases that are suitable for use in a subject method ofregulating production of a target RNA include endoribonucleases thatbind to and cleave a substrate polyribonucleotide in a sequence-specificmanner include enzymatically active polypeptides and that differ from anamino acid sequence set forth in any one of FIGS. 4, 5, and/or 11 byfrom 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20) amino acid substitutions and/or insertions and/ordeletions.

A suitable inducible promoter can include a promoter that is functionalin a eukaryotic cell. Suitable inducible promoters are known in the art.For example, suitable inducible promoters include, but are not limitedto, a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a PHO5promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGKpromoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1.Suitable inducible promoters include tetracycline-inducible promoters; ametallothionein promoter; tetracycline-inducible promoters,methionine-inducible promoters; and galactose-inducible promoters, whichpromoters are all well known in the art. Other suitable promotersinclude the ADH2 alcohol dehydrogenase promoter (repressed in glucose,induced when glucose is exhausted and ethanol is made) and the CUP1metallothionein promoter (induced in the presence of Cu²+, Zn²+).

Agents that induce any given inducible promoter are known in art. Forexample, tetracycline-regulatable promoters can be regulated bytetracycline or doxycycline; carbohydrates can be used to induce acarbohydrate-inducible promoter (e.g., galactose for agalactose-inducible promoter); methionine can be used to induce amethionine-inducible promoter; metals can be used to induce ametallothionein promoter.

The target RNA can be a regulatory RNA. Regulator RNAs are well known inthe art and include, e.g., micro-RNAs, short hairpin RNAs (shRNAs), andthe like.

In some embodiments, cleavage of the target RNA from a precursor RNAinactivates the precursor RNA.

The genetically modified host cell can be an in vitro cell, e.g., aprokaryotic cell, or a eukaryotic cell (e.g., a mammalian cell,including primary cells, transformed cell lines, and the like). Thegenetically modified host cell can be an in vivo cell. In someembodiments, the in vivo cell is a non-human cell.

The genetically modified host cell can be a cell of a multicellularorganism (or can be obtained from a cell of a multicellular organism).

The genetically modified host cell can be a cell obtained from orpresent in an organism of any of the six kingdoms, e.g., Bacteria (e.g.,Eubacteria); Archaebacteria; Protista; Fungi; Plantae; and Animalia.Suitable organisms include plant-like members of the kingdom Protista,including, but not limited to, algae (e.g., green algae, red algae,glaucophytes, cyanobacteria); fungus-like members of Protista, e.g.,slime molds, water molds, etc.; animal-like members of Protista, e.g.,flagellates (e.g., Euglena), amoeboids (e.g., amoeba), sporozoans (e.g,Apicomplexa, Myxozoa, Microsporidia), and ciliates (e.g., Paramecium).Suitable organisms include members of the kingdom Fungi, including, butnot limited to, members of any of the phyla: Basidiomycota (club fungi;e.g., members of Agaricus, Amanita, Boletus, Cantherellus, etc.);Ascomycota (sac fungi, including, e.g., Saccharomyces); Mycophycophyta(lichens); Zygomycota (conjugation fungi); and Deuteromycota. Suitableorganisms include members of the kingdom Plantae, including, but notlimited to, members of any of the following divisions: Bryophyta (e.g.,mosses), Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g.,liverworts), Lycophyta (e.g., club mosses), Sphenophyta (e.g.,horsetails), Psilophyta (e.g., whisk ferns), Ophioglossophyta,Pterophyta (e.g., ferns), Cycadophyta, Gingkophyta, Pinophyta,Gnetophyta, and Magnoliophyta (e.g., flowering plants). Suitableorganisms include members of the kingdom Animalia, including, but notlimited to, members of any of the following phyla: Porifera (sponges);Placozoa; Orthonectida (parasites of marine invertebrates); Rhombozoa;Cnidaria (corals, anemones, jellyfish, sea pens, sea pansies, seawasps); Ctenophora (comb jellies); Platyhelminthes (flatworms);Nemertina (ribbon worms); Ngathostomulida (jawed worms); Gastrotricha;Rotifera; Priapulida; Kinorhyncha; Loricifera; Acanthocephala;Entoprocta; Nemotoda; Nematomorpha; Cycliophora; Mollusca (mollusks);Sipuncula (peanut worms); Annelida (segmented worms); Tardigrada (waterbears); Onychophora (velvet worms); Arthropoda (including the subphyla:Chelicerata, Myriapoda, Hexapoda, and Crustacea, where the Cheliceratainclude, e.g., arachnids, Merostomata, and Pycnogonida, where theMyriapoda include, e.g., Chilopoda (centipedes), Diplopoda (millipedes),Paropoda, and Symphyla, where the Hexapoda include insects, and wherethe Crustacea include shrimp, krill, barnacles, etc.; Phoronida;Ectoprocta (moss animals); Brachiopoda; Echinodermata (e.g. starfish,sea daisies, feather stars, sea urchins, sea cucumbers, brittle stars,brittle baskets, etc.); Chaetognatha (arrow worms); Hemichordata (acornworms); and Chordata. Suitable members of Chordata include any member ofthe following subphyla: Urochordata (sea squirts; including Ascidiacea,Thaliacea, and Larvacea); Cephalochordata (lancelets); Myxini (hagfish);and Vertebrata, where members of Vertebrata include, e.g., members ofPetromyzontida (lampreys), Chondrichthyces (cartilaginous fish),Actinopterygii (ray-finned fish), Actinista (coelocanths), Dipnoi(lungfish), Reptilia (reptiles, e.g., snakes, alligators, crocodiles,lizards, etc.), Aves (birds); and Mammalian (mammals). Suitable plantsinclude any monocotyledon and any dicotyledon.

Thus, e.g., a genetically modified host cell can be a cell obtained fromor present in a protozoan, a plant, a fungus, an algal cell, a yeast, areptile, an amphibian, a mammal, a marine microorganism, a marineinvertebrate, an arthropod, an isopod, an insect, an arachnid, anarchaebacterium, and a eubacterium.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

The genetically modified host cell can be a cell obtained from orpresent in a non-human embryo, e.g., a Drosophila embryo; a zebrafishembryo; a mouse embryo; etc.

The genetically modified host cell can be a stem cell, e.g., an in vitrostem cell; a non-human stem cell; etc. Suitable stem cells includeembryonic stem cells, adult stem cells, and induced pluripotent stem(iPS) cells.

Methods of Isolating a Target Nucleic Acid

The present disclosure provides methods of isolating a target nucleicacid from a mixed population of nucleic acids. The methods generallyinvolve: a) contacting a mixed population of nucleic acids with animmobilized sequence-specific, enzymatically inactive endoribonuclease,wherein the mixed population of nucleic acids includes a target nucleicacid comprising a “tag” (or “recognition”) nucleotide sequence that isspecifically bound by the immobilized sequence-specific, enzymaticallyinactive endoribonuclease, such that the target nucleic acid comprisingthe tag nucleotide sequence (“tagged target nucleic acid”) binds to theimmobilized sequence-specific, enzymatically inactive endoribonuclease,forming a tagged target nucleic acid/immobilized sequence-specificenzymatically active endoribonuclease complex, wherein the contactingstep takes place in a liquid solution (a “binding solution”); and b)adding imidazole to the liquid solution to a final concentration of fromabout 100 mM to about 500 mM (e.g., from about 100 mM to about 150 mM,from about 150 mM to about 200 mM, from about 200 mM to about 250 mM,from about 250 mM to about 300 mM, from about 300 mM to about 350 mM,from about 350 mM to about 400 mM, from about 400 mM to about 450 mM, orfrom about 450 mM to about 500 mM), thereby forming a reactivationsolution that enzymatically reactivates the enzymatically inactiveendoribonuclease such that the endoribonuclease becomes enzymaticallyactive and cleaves the target nucleic acid from the “tag” nucleotidesequence, thereby releasing the target nucleic acid. FIG. 9 is aschematic representation of an exemplary embodiment of a subject methodfor isolating a target RNA.

The method can further include one or more washing steps. For example,after step (a) and before step (b), the immobilized sequence-specific,enzymatically inactive endoribonuclease that comprises a bound targetnucleic acid comprising a “tag” nucleotide sequence can be washed one ormore times with the binding solution, such that the target nucleic acidremains bound to the sequence-specific, enzymatically inactiveendoribonuclease, and any unbound nucleic acids are washed away.

The mixed population of nucleic acids can include RNA and DNA. Thetarget nucleic acid is an RNA that comprises a “tag” or “recognition”nucleotide sequence that is specifically bound by the sequence-specificendoribonuclease. In its enzymatically inactive state (“uninduced”state), the endoribonuclease can bind, but cannot cleave, the taggedtarget RNA. In its enzymatically active state (“induced” state) (e.g.,in the presence of imidazole in a concentration of from about 100 mM toabout 500 mM), the endoribonuclease can both bind and cleave therecognition nucleotide sequence in the tagged target nucleic acid,thereby releasing the target nucleic acid from the tag.

The binding solution can include a buffer and a salt; and lacksimidazole. The reactivation solution can include imidazole in a finalconcentration of from about 100 mM to about 500 mM, e.g., from about 100mM to about 150 mM, from about 150 mM to about 200 mM, from about 250 mMto about 350 mM, from about 350 mM to about 400 mM, or from about 400 mMto about 500 mM. The presence of imidazole reactivates thesequence-specific, enzymatically inactive endoribonuclease such that theendoribonuclease becomes enzymatically active, e.g., theendoribonuclease exhibits at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, or more than 95%, of wild-type sequence-specific endoribonuclease(e.g., an amino acid sequence as depicted in FIG. 5 (e.g., SEQ ID NO:6,8, 9, etc.)). As one non-limiting example, the sequence-specific,enzymatically inactive endoribonuclease is an H29A mutant (orcorresponding variant: see FIG. 4 and FIG. 11) of Csy4 (as describedbelow; and as depicted in FIG. 6 and FIG. 11 (e.g., SEQ ID NO: 105, 106,107, 109, 111, 112)). Contacting the Csy4(H29A) mutant with imidazole,as described above, reactivates the endoribonuclease such that it iscapable of cleaving, in a sequence-specific manner, a recognitionsequence in a target ribonucleic acid. Also suitable for use is an H29A,S50C double mutant of Csy4 (as described below). In some embodiments,the “tag” or recognition sequence comprises the nucleotide sequence5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′ (SEQ ID NO:1) or any of therecognition sequences depicted in FIG. 5 or FIG. 11.

The “tag” or “recognition” nucleotide sequence can be introduced into anucleic acid using standard recombinant methods. Thus, the tagged targetnucleic acid will include a tag that is enzymatically cleaved, therebyreleasing the target nucleic acid.

In some embodiments, the tagged target nucleic acid (RNA) will have oneor more polypeptides bound thereto. A tagged target RNA that has one ormore polypeptides bound thereto is referred to herein as a RNA proteincomplex. Thus, in some embodiments, the target RNA that is isolatedusing a subject method is an RNA protein complex. In some embodiments, asubject method can further comprise analyzing the polypeptide(s) boundto the isolated target RNA.

A subject method provides for isolation of a target RNA (or RNA proteincomplex). In some embodiments, a subject method provides forpurification of a target RNA (or RNA protein complex) such that thetarget RNA (or RNA protein complex) is at least about 50% pure, at leastabout 60% pure, at least about 70% pure, at least about 80% pure, atleast about 90% pure, at least about 95% pure, at least about 98% pure,or greater than 98% pure.

In some embodiments, a protein bound to a target RNA in a targetRNA/protein complex can be eluted from the RNA/protein complex. Theeluted protein can be further characterized, e.g., by sequencing,enzymatic digestion, a functional assay, etc.

The mixed population of nucleic acids can be present in a cell lysate.For example, an expression vector comprising a nucleotide sequenceencoding a tagged target RNA is introduced into a cell (e.g., in vitroor in vivo), such that the cell synthesizes the tagged target RNA. Alysate is made from the cell and the lysate (optionally subjected to oneor more steps to enrich for nucleic acids) is applied to the immobilizedsequence-specific enzymatically-inactive endoribonuclease.

The sequence-specific enzymatically-inactive endoribonuclease can beimmobilized on any of a variety of insoluble support. Suitable insolublesupports include, but are not limited to agarose beads, magnetic beads,a test strip, a multi-well dish, and the like. The insoluble support cancomprise a variety of substances (glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amylose, natural and modified celluloses, polyacrylamides, agaroses, andmagnetite) and can be provided in a variety of forms, including, e.g.,agarose beads, polystyrene beads, latex beads, magnetic beads, colloidmetal particles, glass and/or silicon chips and surfaces, nitrocellulosestrips, nylon membranes, sheets, wells of reaction trays (e.g.,multi-well plates), plastic tubes, etc.

The present disclosure also provides a method of isolating a polypeptidethat binds a target RNA, where the method comprises: a) contacting animmobilized complex with a liquid solution comprising a polypeptide thatbinds the target RNA, where the immobilized complex comprises thevariant Csy4 endoribonuclease and a tagged target RNA comprising arecognition nucleotide sequence that is specifically bound by thevariant Csy4 endoribonuclease, where said contacting results in bindingof the polypeptide to the target RNA, where said contacting is carriedout in a binding solution lacking imidazole; and b) eluting the boundpolypeptide.

Endoribonucleases

The present disclosure provides a sequence-specific endoribonuclease. Insome embodiments, the present disclosure provides a sequence-specificendoribonuclease that binds to a recognition sequence in a targetpolyribonucleotide, but that does not cleave the targetpolyribonucleotide, i.e., the sequence-specific endoribonuclease isenzymatically inactive in hydrolyzing the target polyribonucleotide. Insome embodiments, the present disclosure provides a sequence-specificendoribonuclease that binds to a recognition sequence in a targetpolyribonucleotide, and cleaves the target polyribonucleotide within ornear the recognition sequence, i.e., the sequence-specificendoribonuclease is enzymatically active in hydrolyzing the targetpolyribonucleotide.

In some embodiments, a subject sequence-specific endoribonuclease isimmobilized on an insoluble substrate. Suitable insoluble substratesinclude, but are not limited to agarose beads, magnetic beads, a teststrip, a multi-well dish, and the like. The insoluble substrate cancomprise a variety of substances (glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amylose, natural and modified celluloses, polyacrylamides, agaroses, andmagnetite) and can be provided in a variety of forms, including, e.g.,agarose beads, polystyrene beads, latex beads, magnetic beads, colloidmetal particles, glass and/or silicon chips and surfaces, nitrocellulosestrips, nylon membranes, sheets, wells of reaction trays (e.g.,multi-well plates), plastic tubes, etc.

Enzymatically Inactive Sequence-Specific Endoribonuclease

The present disclosure provides an enzymatically inactive,sequence-specific endoribonuclease, wherein the enzymatically inactivesequence-specific endoribonuclease binds to a target sequence in apolyribonucleotide in a sequence-specific manner. A subjectenzymatically inactive, sequence-specific endoribonuclease binds atarget polyribonucleotide in a sequence-specific manner, but does notcleave the target polyribonucleotide. A subject enzymatically inactive,sequence-specific endoribonuclease is useful for isolating a target RNAfrom a mixed population of nucleic acids, as described above.

In some embodiments, a subject enzymatically inactive, sequence-specificendoribonuclease comprises one or more amino acid substitutions comparedto a naturally-occurring, enzymatically active, Csy4, CasE, or Cas6polypeptide.

In some embodiments, a subject enzymatically inactive, sequence-specificendoribonuclease comprises an amino acid substitution at His-29 of aCsy4 polypeptide, or at an equivalent position in a CasE or a Cas6polypeptide. In some embodiments, a subject enzymatically inactive,sequence-specific endoribonuclease comprises an amino acid substitutionat Ser-148 of a Csy4 polypeptide, or at an equivalent position in a CasEor a Cas6 polypeptide.

FIG. 6 (SEQ ID NO: 101, 102) and FIG. 11 (SEQ ID NO: 105, 106, 107, 109,111, 112) depict non-limiting examples of suitable enzymaticallyinactive, sequence-specific endoribonuclease amino acid sequences. Insome embodiments, a subject enzymatically inactive, sequence-specificendoribonuclease comprises an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity with an amino acid sequence depicted in FIG. 6(SEQ ID NO: 101, 102) or FIG. 11 (SEQ ID NO: 105, 106, 107, 109, 111,112), where the amino acid sequence includes a substitution at His-29,Ser-50, or both His-29 and Ser-50. For example, the variant Csy4endoribonuclease can include a H29A (His-29 to Ala-29) substitution (ora corresponding His to Ala variant according to sequence alignment,e.g., FIG. 4, such as H34A, His-34 to Ala-34, in SEQ ID NO: 108, 109(see FIG. 11D)), a S50C (Ser-50 to Cys-50) substitution, or both a H29Aand a S50C substitution.

In some embodiments, a subject enzymatically inactive, sequence-specificendoribonuclease is a variant Csy4 endoribonuclease. In some cases, asubject variant Csy4 endoribonuclease comprises an amino acid sequencehaving at least about 95% amino acid sequence identity to the amino acidsequence set forth in FIG. 6, where the endoribonuclease comprises anamino acid substitution at His-29, where the variant Csy4endoribonuclease is enzymatically inactive in the absence of imidazole,and where the variant Csy4 endoribonuclease is activatable in thepresence of imidazole. In some instances, the amino acid substitution isa His29 to Ala29 substitution. In some cases, variant Csy4endoribonuclease also includes a Ser-50 substitution. In some instances,a subject variant Csy4 endoribonuclease binds an RNA substrate thatcomprises the nucleotide sequence 5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′(SEQ ID NO:1) or any of the recognition sequences depicted in FIG. 5 orFIG. 11.

A subject enzymatically inactive, sequence-specific endoribonuclease is“conditionally” enzymatically inactive, e.g., a subject enzymaticallyinactive, sequence-specific endoribonuclease (e.g., a subject variantCsy4 endoribonuclease) is enzymatically inactive in the absence ofimidazole; and the enzymatically inactive, sequence-specificendoribonuclease (e.g., subject variant Csy4 endoribonuclease) isactivatable by imidazole. For example, the enzymatically inactive,sequence-specific endoribonuclease (e.g., subject variant Csy4endoribonuclease) can be enzymatically activated by contacting theendoribonuclease with imidazole at a concentration of from about 100 mMto about 500 mM.

The presence of imidazole (e.g., in a concentration range of from about100 mM to about 500 mM) reactivates the sequence-specific, enzymaticallyinactive endoribonuclease such that the endoribonuclease becomesenzymatically active, e.g., the endoribonuclease exhibits at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, or more than 95%, of wild-typesequence-specific endoribonuclease (e.g., an amino acid sequence asdepicted in FIG. 5 (e.g., SEQ ID NO:6, 8, or 9) or FIG. 11 (SEQ ID NO:39, 79, 84, 90, 104, 108, or 110)).

In some embodiments, a subject enzymatically inactive, sequence-specificendoribonuclease (e.g., a subject variant Csy4 endoribonuclease)comprises a detectable label, including a moiety that provides adetectable signal. Suitable detectable labels and/or moieties thatprovide a detectable signal include, but are not limited to, an enzyme,a radioisotope, a member of a FRET pair, a member of a specific bindingpair; a fluorophore; a fluorescent protein; a quantum dot; and the like.

FRET pairs (donor/acceptor) suitable for use include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL,fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5 andfluorescein/LC Red 705. In addition, a fluorophore/quantum dotdonor/acceptor pair can be used.

Suitable fluorophores (“fluorescent label”) include any molecule thatmay be detected via its inherent fluorescent properties, which includefluorescence detectable upon excitation. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 andOregon green. Suitable optical dyes are described in the 2002 MolecularProbes Handbook, 9th Ed., by Richard P. Haugland, hereby expresslyincorporated by reference.

Suitable enzymes include, but are not limited to, horse radishperoxidase, luciferase, β-galactosidase, and the like.

Suitable fluorescent proteins include, but are not limited to, a greenfluorescent protein (GFP), e.g., a GFP from Aequoria victoria or amutant or derivative thereof e.g., as described in U.S. Pat. Nos.6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750; 5,968,738;5,958,713; 5,919,445; 5,874,304; a red fluorescent protein; a yellowfluorescent protein; any of a variety of fluorescent and coloredproteins from Anthozoan species, as described in, e.g., Matz et al.(1999) Nature Biotechnol. 17:969-973; and the like.

Suitable nanoparticles include, e.g., quantum dots (QDs), fluorescent orluminescent nanoparticles, and magnetic nanoparticles. Any optical ormagnetic property or characteristic of the nanoparticle(s) can bedetected.

QDs and methods for their synthesis are well known in the art (see,e.g., U.S. Pat. Nos. 6,322,901; 6,576,291; and 6,815,064). QDs can berendered water soluble by applying coating layers comprising a varietyof different materials (see, e.g., U.S. Pat. Nos. 6,423,551; 6,251,303;6,319,426; 6,426,513; 6,444,143; and 6,649,138). For example, QDs can besolubilized using amphiphilic polymers. Exemplary polymers that havebeen employed include octylamine-modified low molecular weightpolyacrylic acid, polyethylene-glycol (PEG)-derivatized phospholipids,polyanhydrides, block copolymers, etc. QDs can be conjugated to apolypeptide via any of a number of different functional groups orlinking agents that can be directly or indirectly linked to a coatinglayer (see, e.g., U.S. Pat. Nos. 5,990,479; 6,207,392; 6,251,303;6,306,610; 6,325,144; and 6,423,551).

QDs with a wide variety of absorption and emission spectra arecommercially available, e.g., from Quantum Dot Corp. (Hayward Calif.;now owned by Invitrogen) or from Evident Technologies (Troy, N.Y.). Forexample, QDs having peak emission wavelengths of approximately 525, 535,545, 565, 585, 605, 655, 705, and 800 nm are available. Thus the QDs canhave a range of different colors across the visible portion of thespectrum and in some cases even beyond.

Suitable radioisotopes include, but are not limited to ¹⁴C, ³H, ³²P,³³P, ³⁵S, ¹²⁵I, and ¹³¹I. The use of radioisotopes as labels is wellknown in the art.

In some embodiments, a subject enzymatically inactive, sequence-specificendoribonuclease (e.g., a subject variant Csy4 endoribonuclease) isimmobilized on an insoluble substrate. Suitable insoluble substratesinclude, but are not limited to agarose beads, magnetic beads, a teststrip, a multi-well dish, and the like. The insoluble substrate cancomprise a variety of substances (glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amylose, natural and modified celluloses, polyacrylamides, agaroses, andmagnetite) and can be provided in a variety of forms, including, e.g.,agarose beads, polystyrene beads, latex beads, magnetic beads, colloidmetal particles, glass and/or silicon chips and surfaces, nitrocellulosestrips, nylon membranes, sheets, wells of reaction trays (e.g.,multi-well plates), plastic tubes, etc.

In some embodiments, a subject enzymatically inactive, sequence-specificendoribonuclease (e.g., a subject variant Csy4 endoribonuclease) ispurified, e.g., is at least 80% pure, at least 85% pure, at least 90%pure, at least 95% pure, at least 98% pure, at least 99% pure, orgreater than 99% pure.

Compositions

The present disclosure provides compositions comprising a subjectsequence-specific, enzymatically inactive, endoribonuclease. A subjectcomposition can comprise, in addition to a subject sequence-specific,enzymatically inactive, endoribonuclease, one or more of: a salt, e.g.,NaCl, MgCl, KCl, MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; and the like.

Enzymatically Active Sequence-Specific Endoribonuclease

In some embodiments, a subject enzymatically active sequence-specificendoribonuclease comprises a moiety that provides for detection. Forexample, a subject enzymatically active sequence-specificendoribonuclease can comprise a covalently or non-covalently linkedmoiety that provides for detection.

Suitable detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Moieties that provide for detection include,but are not limited to, a fluorescent molecule; a quantum dot; an enzyme(other than the endoribonuclease), where the enzyme catalyzes conversionof a substrate to a detectable product, where the product is directlydetectable; a nanoparticle; and the like.

Suitable fluorescent proteins that can be linked to a subjectenzymatically active sequence-specific endoribonuclease include, but arenot limited to, a green fluorescent protein (GFP), e.g., a GFP fromAequoria victoria or a mutant or derivative thereof e.g., as describedin U.S. Pat. Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750;5,968,738; 5,958,713; 5,919,445; 5,874,304; a red fluorescent protein; ayellow fluorescent protein; any of a variety of fluorescent and coloredproteins from Anthozoan species, as described in, e.g., Matz et al.(1999) Nature Biotechnol. 17:969-973; and the like.

Suitable nanoparticles include, e.g., quantum dots (QDs), fluorescent orluminescent nanoparticles, and magnetic nanoparticles. Any optical ormagnetic property or characteristic of the nanoparticle(s) can bedetected.

QDs and methods for their synthesis are well known in the art (see,e.g., U.S. Pat. Nos. 6,322,901; 6,576,291; and 6,815,064). QDs can berendered water soluble by applying coating layers comprising a varietyof different materials (see, e.g., U.S. Pat. Nos. 6,423,551; 6,251,303;6,319,426; 6,426,513; 6,444,143; and 6,649,138). For example, QDs can besolubilized using amphiphilic polymers. Exemplary polymers that havebeen employed include octylamine-modified low molecular weightpolyacrylic acid, polyethylene-glycol (PEG)-derivatized phospholipids,polyanhydrides, block copolymers, etc. QDs can be conjugated to apolypeptide via any of a number of different functional groups orlinking agents that can be directly or indirectly linked to a coatinglayer (see, e.g., U.S. Pat. Nos. 5,990,479; 6,207,392; 6,251,303;6,306,610; 6,325,144; and 6,423,551).

QDs with a wide variety of absorption and emission spectra arecommercially available, e.g., from Quantum Dot Corp. (Hayward Calif.;now owned by Invitrogen) or from Evident Technologies (Troy, N.Y.). Forexample, QDs having peak emission wavelengths of approximately 525, 535,545, 565, 585, 605, 655, 705, and 800 nm are available. Thus the QDs canhave a range of different colors across the visible portion of thespectrum and in some cases even beyond.

In some embodiments, a subject enzymatically active, sequence-specificendoribonuclease is purified, e.g., is at least 80% pure, at least 85%pure, at least 90% pure, at least 95% pure, at least 98% pure, at least99% pure, or greater than 99% pure.

Compositions

The present disclosure provides compositions comprising a subjectsequence-specific, enzymatically active endoribonuclease. A subjectcomposition can comprise, in addition to a subject sequence-specificenzymatically active, endoribonuclease, one or more of: a salt, e.g.,NaCl, MgCl, KCl, MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; and the like.

The present disclosure provides compositions comprising a subjectsequence-specific, enzymatically inactive endoribonuclease (e.g., asubject variant Csy4 endoribonuclease). A subject composition cancomprise, in addition to a subject sequence-specific enzymaticallyinactive endoribonuclease (e.g., a subject variant Csy4endoribonuclease), one or more of: a salt, e.g., NaCl, MgCl, KCl, MgSO₄,etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; and the like. In some embodiments,the composition lacks imidazole. In some embodiments, the compositioncomprises imidazole in a concentration of from about 100 mM to about 500mM.

Methods of Producing a Subject Sequence-Specific Endoribonuclease

A subject sequence-specific endoribonuclease (e.g., a subjectsequence-specific enzymatically active, endoribonuclease; a subjectsequence-specific enzymatically inactive, endoribonuclease) can beproduced by any known method, e.g., conventional synthetic methods forprotein synthesis; recombinant DNA methods; etc.

Where a subject sequence-specific endoribonuclease is chemicallysynthesized, the synthesis may proceed via liquid-phase or solid-phase.Solid phase polypeptide synthesis (SPPS), in which the C-terminal aminoacid of the sequence is attached to an insoluble support followed bysequential addition of the remaining amino acids in the sequence, is anexample of a suitable method for the chemical synthesis of a subjectsequence-specific endoribonuclease. Various forms of SPPS, such as Fmocand Boc, are available for synthesizing a subject sequence-specificendoribonuclease. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85:2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed.Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev.Med Chem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett.12:723-8.

Standard recombinant methods can be used for production of a subjectsequence-specific endoribonuclease. For example, nucleic acids encodinga subject sequence-specific endoribonuclease are inserted intoexpression vectors. The DNA segments encoding a subjectsequence-specific endoribonuclease are operably linked to controlsequences in the expression vector(s) that ensure the expression of theencoded polypeptides. Expression control sequences include, but are notlimited to, promoters (e.g., naturally-associated or heterologouspromoters), signal sequences, enhancer elements, and transcriptiontermination sequences. The expression control sequences can beeukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells (e.g., COS or CHO cells). Once thevector has been incorporated into the appropriate host, the host ismaintained under conditions suitable for high level expression of thenucleotide sequences, and the collection and purification of theendoribonuclease.

Nucleic Acids and Host Cells

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a subject sequence-specific endoribonuclease (e.g., asubject sequence-specific, enzymatically active endoribonuclease; asubject sequence-specific, enzymatically inactive endoribonuclease). Insome embodiments, the nucleic acid is an expression vector, where theexpression vector can provide for production of the sequence-specificendoribonuclease, e.g., in a cell.

A nucleotide sequence encoding a subject sequence-specificendoribonuclease (e.g., a subject sequence-specific, enzymaticallyactive endoribonuclease; a subject sequence-specific, enzymaticallyinactive endoribonuclease) can be operably linked to one or moreregulatory elements, such as a promoter and enhancer, that allowexpression of the nucleotide sequence in the intended target cells(e.g., a cell that is genetically modified to synthesize the encodedendoribonuclease).

In some embodiments, a subject nucleic acid comprises a nucleotidesequence encoding a polypeptide having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, with an amino acidsequence set forth in FIG. 4, FIG. 5, and/or FIG. 11. In someembodiments, a subject nucleic acid comprises a nucleotide sequenceencoding a variant Csy4 polypeptide, as described above.

A nucleotide sequence encoding a subject sequence-specificendoribonuclease (e.g., a subject sequence-specific, enzymaticallyactive endoribonuclease; a subject sequence-specific, enzymaticallyinactive endoribonuclease) can be operably linked to a transcriptioncontrol element (e.g., a promoter, an enhancer, etc.). Suitable promoterand enhancer elements are known in the art. For expression in abacterial cell, suitable promoters include, but are not limited to,lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in aeukaryotic cell, suitable promoters include, but are not limited to,cytomegalovirus immediate early promoter; herpes simplex virus thymidinekinase promoter; early and late SV40 promoters; promoter present in longterminal repeats from a retrovirus; mouse metallothionein-I promoter;and various art-known tissue specific promoters.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spy promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda). Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the Lad repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

A nucleotide sequence encoding a subject sequence-specificendoribonuclease (e.g., a subject sequence-specific, enzymaticallyactive endoribonuclease; a subject sequence-specific, enzymaticallyinactive endoribonuclease) can be present in an expression vector and/ora cloning vector. An expression vector can include a selectable marker,an origin of replication, and other features that provide forreplication and/or maintenance of the vector.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant construct. The following vectors are provided by wayof example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Suitable expression vectorsinclude, but are not limited to, viral vectors (e.g. viral vectors basedon vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., InvestOpthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., HGene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see,e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al.,Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594,1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989)63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte etal., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; humanimmunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23,1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector(e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derivedfrom retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, human immunodeficiency virus, myeloproliferativesarcoma virus, and mammary tumor virus); and the like.

The present disclosure provides isolated genetically modified host cells(e.g., in vitro cells) that are genetically modified with a subjectnucleic acid. In some embodiments, a subject isolated geneticallymodified host cell can produce a subject sequence-specificendoribonuclease (e.g., a subject sequence-specific, enzymaticallyactive endoribonuclease; a subject sequence-specific, enzymaticallyinactive endoribonuclease).

Suitable host cells include eukaryotic host cells, such as a mammaliancell, an insect host cell, a yeast cell; and prokaryotic cells, such asa bacterial cell. Introduction of a subject nucleic acid into the hostcell can be effected, for example by calcium phosphate precipitation,DEAE dextran mediated transfection, liposome-mediated transfection,electroporation, or other known method.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

Suitable yeast cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302. Examples of Salmonella strains whichcan be employed in the present invention include, but are not limitedto, Salmonella typhi and S. typhimurium. Suitable Shigella strainsinclude, but are not limited to, Shigella flexneri, Shigella sonnei, andShigella disenteriae. Typically, the laboratory strain is one that isnon-pathogenic. Non-limiting examples of other suitable bacteriainclude, but are not limited to, Bacillus subtilis, Pseudomonas pudita,Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides,Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and thelike. In some embodiments, the host cell is Escherichia coli.

Kits

The present disclosure also provides kits for determining the nucleotidesequence of a target polyribonucleotide. The present disclosure provideskits for carrying out sequence-specific cleavage of a substratepolyribonucleotide. The present disclosure provides kits for carryingout detection of an RNA sequence in a target polyribonucleotide. Thepresent disclosure provides kits for carrying out isolation of a targetRNA. The present disclosure provides kits for carrying out isolation ofa polypeptide that binds a target RNA.

Kits for Carrying Out Direct Sequencing of a Polyribonucleotide

A subject kit for carrying out direct sequencing of a polyribonucleotideincludes at least a subject sequence-specific, enzymatically inactiveendoribonuclease, where the sequence-specific, enzymatically inactiveendoribonuclease is purified. In some embodiments, the enzymaticallyinactive, sequence-specific endoribonuclease is linked to an acceptormolecule or a donor molecule, for FRET detection.

A subject kit for carrying out direct sequencing of a polyribonucleotideincludes at least a subject sequence-specific, enzymatically inactiveendoribonuclease; and can include one or more additional components,where the one or more additional components can be: 1) a buffer; 2) aprobe oligonucleotide comprising a defined sequence; 3) a probeoligonucleotide comprising a defined sequence, where the probeoligonucleotide is linked to an acceptor molecule or a donor molecule,for FRET detection; 4) an insoluble support, for linking to a targetpolyribonucleotide; 5) a positive control polyribonucleotide, where thepositive control polyribonucleotide comprises a known nucleotidesequence; 6) a positive control probe oligonucleotide that binds to andforms a duplex with the known sequence of the positive controlpolyribonucleotide.

In addition to above-mentioned components, a subject kit can furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Kits for Carrying Out Sequence-Specific Cleavage of a SubstratePolyribonucleotide

A subject kit for carrying out sequence-specific cleavage of a substratepolyribonucleotide includes at least a purified sequence-specificendoribonuclease and/or a nucleic acid comprising a nucleotide sequenceencoding the sequence-specific endoribonuclease. A subject kit forcarrying out sequence-specific cleavage of a substratepolyribonucleotide can include, in addition to a purifiedsequence-specific endoribonuclease (and/or a nucleic acid comprising anucleotide sequence encoding the sequence-specific endoribonuclease),one or more additional components. Suitable additional componentsinclude, e.g., a buffer; a polyribonucleotide substrate that serves as apositive control; polyribonucleotide size standards; a negative controlsubstrate; and the like. The components can each be in separatecontainers. The kit can further include one or more positive andnegative controls.

In addition to above-mentioned components, a subject kit can furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Kits for Carrying Out Detection of a Sequence in a TargetPolyribonucleotide

A subject kit for carrying out detection of a sequence in a targetpolyribonucleotide (e.g., for carrying out detection of apolyribonucleotide) can include an oligonucleotide probe comprising aknown sequence. In some embodiments, the kit will include anoligonucleotide probe comprising a known sequence and comprising adetectable moiety, e.g., a polypeptide that can be detected using animmunological assay; a fluorescent protein; a luciferin; etc. The kitcan further include a positive control polyribonucleotide that comprisesa nucleotide sequence capable of forming a duplex with theoligonucleotide probe. The kit can further include an enzymaticallyactive, sequence-specific endoribonuclease that specifically detects andcleaves a duplex formed by the oligonucleotide probe and a targetpolyribonucleotide. The kit can further include one or more of a buffer;components for detecting the detectable moiety; a test strip; and thelike. The kit can further include one or more positive and negativecontrols.

In addition to above-mentioned components, a subject kit can furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Kits for Carrying Out Isolation of a Target RNA

A subject kit for carrying out isolation (e.g., purification) of atarget RNA can include one or more of: 1) a subject sequence-specific,enzymatically inactive endoribonuclease; 2) an expression constructcomprising a “tag” nucleotide sequence, i.e., a nucleotide sequence thatis specifically bound by the sequence-specific, enzymatically inactiveendoribonuclease, where a nucleotide sequence encoding a target RNA ofchoice can be inserted 3′ of the “tag” nucleotide sequence; and 3)imidazole. The sequence-specific, enzymatically inactiveendoribonuclease can be immobilized on an insoluble support. The kit canfurther include a liquid composition for contacting a mixed populationof nucleic acids with the immobilized sequence-specific, enzymaticallyinactive endoribonuclease. The kit can further include a wash buffer.The kit can further include one or more positive and negative controls.A positive control could include an expression vector comprising anucleotide sequence encoding a tagged target RNA, where the tag isspecifically bound by the sequence-specific, enzymatically inactiveendoribonuclease. The components can each be in separate containers.

For example, a subject kit can include a subject sequence-specific,enzymatically inactive endoribonuclease. A subject kit can furtherinclude a recombinant expression vector comprising, in order from 5′ to3′ and in operable linkage: a) a nucleotide sequence encoding an RNAsubstrate that is specifically bound by a subject variant Csy4endoribonuclease; and b) a multiple cloning site suitable for insertionof a nucleic acid encoding the target RNA. The nucleotide sequenceencoding the RNA substrate can be operably linked to a promoter. In someinstances, the promoter is an inducible promoter. The RNA substrate cancomprise the nucleotide sequence 5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′ (SEQID NO:1) or any of the recognition sequences depicted in FIG. 5 or FIG.11. In some cases, the recombinant expression vector comprises, insertedinto the multiple cloning site, a nucleotide sequence encoding thetarget RNA. The kit can further include a buffer that lacks imidazole.The kit can further include imidazole or an imidazole solution. The kitcan further include one or more wash buffers. In some cases, the kitwill include a positive control expression vector. The variant Csy4endoribonuclease can be immobilized on an insoluble support, wheresuitable insoluble supports include, but are not limited to agarosebeads, magnetic beads, a test strip, a multi-well dish, and the like.The insoluble support can comprise a variety of substances (glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amylose, natural and modified celluloses,polyacrylamides, agaroses, and magnetite) and can be provided in avariety of forms, including, e.g., agarose beads, polystyrene beads,latex beads, magnetic beads, colloid metal particles, glass and/orsilicon chips and surfaces, nitrocellulose strips, nylon membranes,sheets, wells of reaction trays (e.g., multi-well plates), plastictubes, etc.

In addition to above-mentioned components, a subject kit can furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Methods of Directly Sequencing a Target Polyribonucleotide

The present disclosure provides a method of directly determining thenucleotide sequence of a target polyribonucleotide. Thus, for example,the method does not require synthesis of a polydeoxyribonucleotidecounterpart of a target polyribonucleotide in order to determine thenucleotide sequence of the target polyribonucleotide.

Viral diagnostics, personalized medicine, single-cell transcriptanalysis, and translational profiling are all fields in which direct RNAdetection and sequencing find use. A subject polyribonucleotidesequencing method, and a subject method of detecting a specific sequencein a polyribonucleotide, find use in these various fields.

A subject polyribonucleotide sequencing method generally involves: a)contacting a target polyribonucleotide with an oligonucleotide probecomprising a specific known sequence and an enzymatically inactivesequence-specific endoribonuclease under conditions that favor duplexformation between the oligonucleotide probe and the targetpolyribonucleotide, wherein the enzymatically inactive sequence-specificendoribonuclease binds the specific sequence in the duplex; and b)detecting specific binding between the oligonucleotide probe and thetarget polyribonucleotide, where specific binding of the enzymaticallyinactive sequence-specific endoribonuclease to the duplex indicates thepresence of the specific sequence in the target polyribonucleotide.

In some cases, the enzymatically inactive sequence-specificendoribonuclease is linked (covalently or non-covalently) to an emissivelabel. By “emissive label” is meant any molecule that may be detectedvia its inherent emission properties, which include emission detectableupon excitation. Suitable emissive labels include, but are not limitedto, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640,Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes aredescribed in the 2002 Molecular Probes Handbook, 9th Ed., by Richard P.Haugland.

In some instances, the oligonucleotide probe used in a subjectpolyribonucleotide sequencing method is linked to a donor molecule, theenzymatically inactive sequence-specific endoribonuclease is linked toan acceptor molecule, and detection of duplex formation is byfluorescence resonance energy transfer (also referred to as “Försterresonance energy transfer” or “FRET”).

Förster resonance energy transfer (FRET) is phenomenon known in the artwherein excitation of one emissive dye is transferred to another withoutemission of a photon. A FRET pair consists of a donor chromophore and anacceptor chromophore (where the acceptor chromophore may be a quenchermolecule). The emission spectrum of the donor and the absorptionspectrum of the acceptor must overlap, and the two molecules must be inclose proximity. The distance between donor and acceptor at which 50% ofdonors are deactivated (transfer energy to the acceptor) is defined bythe Förster radius, which is typically 10-100 angstroms. Changes in theemission spectrum comprising FRET pairs can be detected, indicatingchanges in the number of that are in close proximity (i.e., within 100angstroms of each other). This will typically result from the binding ordissociation of two molecules, one of which is labeled with a FRET donorand the other of which is labeled with a FRET acceptor, wherein suchbinding brings the FRET pair in close proximity.

Binding of such molecules will result in an increased emission of theacceptor and/or quenching of the fluorescence emission of the donor.FRET pairs (donor/acceptor) suitable for use include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL,fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5 andfluorescein/LC Red 705. In addition, a fluorophore/quantum dotdonor/acceptor pair can be used. EDANS is(5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid);DABCYL is 4-(4-dimethylaminophenyl) diazenylbenzoic acid.

Cy3, Cy5, Cy 5.5, and the like, are cyanines. For example, Cy3 and Cy5are reactive water-soluble fluorescent dyes of the cyanine dye family.Cy3 dyes are red (˜550 nm excitation, ˜570 nm emission and thereforeappear green), while Cy5 is fluorescent in the red region (˜650/670 nm)but absorbs in the orange region (˜649 nm). Alexa Fluor dyes, Dylight,IRIS Dyes, Seta dyes, SeTau dyes, SRfluor dyes and Square dyes can alsobe used.

In another aspect of FRET, an emissive donor molecule and a nonemissiveacceptor molecule (“quencher”) may be employed. In this application,emission of the donor will increase when quencher is displaced fromclose proximity to the donor and emission will decrease when thequencher is brought into close proximity to the donor. Useful quenchersinclude, but are not limited to, DABCYL, QSY 7 and QSY 33. Usefulfluorescent donor/quencher pairs include, but are not limited toEDANS/DABCYL, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL,coumarin/DABCYL and fluorescein/QSY 7 dye.

In some cases, the enzymatically inactive sequence-specificendoribonuclease is linked (covalently or non-covalently) to a labelenzyme. By “label enzyme” is meant an enzyme which may be reacted in thepresence of a label enzyme substrate which produces a detectableproduct. Suitable label enzymes also include optically detectable labels(e.g., in the case of horse radish peroxidase (HRP)). Suitable labelenzymes include but are not limited to, HRP, alkaline phosphatase,luciferase, β-galactosidase, and glucose oxidase. Methods for the use ofsuch substrates are well known in the art. The presence of the labelenzyme is generally revealed through the enzyme's catalysis of areaction with a label enzyme substrate, producing an identifiableproduct. Such products may be opaque, such as the reaction ofhorseradish peroxidase with tetramethyl benzedine, and may have avariety of colors. Other label enzyme substrates, such as Luminol(available from Pierce Chemical Co.), have been developed that producefluorescent reaction products. Methods for identifying label enzymeswith label enzyme substrates are well known in the art and manycommercial kits are available. Examples and methods for the use ofvarious label enzymes are described in Savage et al., Previews 247:6-9(1998), Young, J. Virol. Methods 24:227-236 (1989).

In some cases, the enzymatically inactive sequence-specificendoribonuclease comprises a radioisotope. By “radioisotope” is meantany radioactive molecule. Suitable radioisotopes for use in theinvention include, but are not limited to ¹⁴C, ³H, ³²P, ³³P, ³⁵S, ¹²⁵I,and ¹³¹I. The use of radioisotopes as labels is well known in the art.

In some cases, the enzymatically inactive sequence-specificendoribonuclease is linked (covalently or non-covalently) to a member ofa specific binding pair (“partner of a binding pair”). By “partner of abinding pair” or “member of a binding pair” is meant one of a first anda second moiety, wherein the first and the second moiety have a specificbinding affinity for each other. Suitable binding pairs include, but arenot limited to, antigen/antibodies (for example,digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP,dansyl-X-anti-dansyl, fluorescein/anti-fluorescein, luciferyellow/anti-lucifer yellow, and rhodamine anti-rhodamine), biotin/avidin(or biotin/streptavidin) and calmodulin binding protein(CBP)/calmodulin.

In some embodiments, the oligonucleotide probe comprises a modificationthat provides for increased resistance to non-specific hydrolysis. Suchmodifications are well known in the art and include, e.g.,nuclease-resistant internucleosidic linkages, modified backbones, basemodifications, base substitutions, sugar modifications, and the like.

Suitable modified oligonucleotide backbones containing a phosphorus atomtherein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be a basic (the nucleobase is missing or has ahydroxyl group in place thereof). Various salts (such as, for example,potassium or sodium), mixed salts and free acid forms are also included.

A modified oligonucleotide can comprise one or more phosphorothioateand/or heteroatom internucleoside linkages, in particular—CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene (methylimino)or MMI backbone), —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—O—N(CH₃)—CH₂—CH₂— (wherein the native phosphodiester internucleotidelinkage is represented as —O—P(═O)(OH)—O—CH₂—). MMI type internucleosidelinkages are disclosed in the above referenced U.S. Pat. No. 5,489,677.Suitable amide internucleoside linkages are disclosed in t U.S. Pat. No.5,602,240.

A modified oligonucleotide can comprise one or more morpholino backbonestructures as described in, e.g., U.S. Pat. No. 5,034,506. For example,in some embodiments, a modified oligonucleotide comprises a 6-memberedmorpholino ring in place of a ribose ring. In some of these embodiments,a phosphorodiamidate or other non-phosphodiester internucleoside linkagereplaces a phosphodiester linkage. Morpholino nucleic acids(“morpholinos”) include bases bound to morpholine rings instead ofdeoxyribose rings; in addition, the phosphate backbone can include anon-phosphate group, e.g., a phosphorodiamidate group instead ofphosphates. Summerton (1999) Biochim. Biophys. Acta 1489:141; Heasman(2002) Dev. Biol. 243:209; Summerton and Weller (1997) Antisense & Nucl.Acid Drug Dev. 7:187; Hudziak et al. (1996) Antisense & Nucl. Acid DrugDev. 6:267; Partridge et al. (1996) Antisense & Nucl. Acid Drug Dev.6:169; Amantana et al. (2007) Bioconj. Chem. 18:1325; Morcos et al.(2008) BioTechniques 45:616.

A modified oligonucleotide can comprise a modified backbone. Modifiedpolynucleotide backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

A modified oligonucleotide can comprise one or more substituted sugarmoieties. Suitable oligonucleotides comprise a sugar substituent groupselected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Also suitable are O((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Othersuitable oligonucleotides comprise a sugar substituent group selectedfrom: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, and the like.A suitable modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further suitablemodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (alsoknown in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE),i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂.

A modified oligonucleotide can comprise one or more nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.

A suitable enzymatically inactive sequence-specific endoribonucleaseincludes an enzymatically inactive sequence-specific endoribonucleasedescribed hereinbelow. For example, an enzymatically inactivesequence-specific endoribonuclease as depicted in FIG. 6 can be used.

In some embodiments, the target polyribonucleotide is linked (covalentlyor non-covalently) to a solid support (an insoluble support). Suitableinsoluble supports include, but are not limited to, beads, plates (e.g.,multi-well plates), strips, etc., where the insoluble support cancomprise various materials including, but not limited to, polystyrene,polypropylene, agarose, and the like.

Oligonucleotide probes (“detection oligonucleotide”) can be RNA, DNA, orany chemically modified version of an RNA or DNA, e.g., peptide nucleicacids (PNAs), locked nucleic acids (LNAs), and the like.

A subject polyribonucleotide sequencing method can include one or morewashing steps, e.g., to remove non-specifically bound components such asnon-specifically bound oligonucleotide probes, any non-specificallybound detectable moieties, and the like.

A non-limiting example of how to carry out a subject polyribonucleotidesequencing method is as follows. A target polyribonucleotide bound to asolid support. The target polyribonucleotide is of unknown sequence andis the “RNA to be sequenced.” Four oligonucleotide probes of fourdifferent known nucleotide sequences each comprise a differentfluorophore (fluorophores 1-4). The fluorophores are members of FRETpairs. The counterpart members of the FRET pairs are quantum dots. Thequantum dot is linked to an enzymatically inactive sequence-specificendoribonuclease. The enzymatically inactive sequence-specificendoribonuclease binds, but does not cleave, the duplex formed betweenan oligonucleotide probe and the target polyribonucleotide. Only one ofthe four oligonucleotide probes binds to and forms a duplex with thetarget polyribonucleotide. A washing step removes any unboundoligonucleotide probes. Binding of oligonucleotide probe-fluorophore2results in duplex formation with the target polyribonucleotide.Fluorophore2 is thus brought into proximity to the quantum dot linked tothe enzymatically inactive sequence-specific endoribonuclease, andfluorescence is quenched.

Methods of Cleaving a Polyribonucleotide

The present disclosure provides a method of cleaving apolyribonucleotide in a sequence-specific manner. The method generallyinvolves contacting a substrate polyribonucleotide with an enzymaticallyactive sequence-specific endoribonuclease (e.g., a Csy4endoribonuclease) under conditions that favor sequence-specific cleavageof the polyribonucleotide substrate. A subject method of cleaving apolyribonucleotide in a sequence-specific manner can be used to: 1)remove an affinity tag from a substrate polyribonucleotide; 2) togenerate a population of product polyribonucleotides having homogeneityat the 5′ end, e.g., where the substrate polyribonucleotides are invitro transcribed mRNAs; and 3) to regulate gene expression in a cell invitro or in vivo.

Substrate Polyribonucleotides

The terms “substrate polyribonucleotide” and “target polyribonucleotide”are used interchangeably herein to refer to a polyribonucleotide that isbound by a sequence-specific endoribonuclease in a sequence-specificmanner. A substrate polyribonucleotide can be single stranded. In someinstances, a substrate polyribonucleotide is double stranded.

An endoribonuclease binds to and cleaves a substrate polyribonucleotidein a sequence-specific manner. Thus, for example, an endoribonucleasebinds to and cleaves a substrate polyribonucleotide at a specificsequence, referred to herein as a “recognition sequence” or a“recognition site.”

A recognition sequence can be a tetranucleotide sequence, apentanucleotide sequence, a hexanucleotide sequence, a heptanucleotidesequence, an octanucleotide sequence, or longer than an octanucleotide.For example, in some embodiments, the recognition sequence is 9ribonucleotides, 10 ribonucleotides, 11 ribonucleotides, 12ribonucleotides, 13 ribonucleotides, 14 ribonucleotides, 15ribonucleotides, 16 ribonucleotides, 17 ribonucleotides, 18ribonucleotides, 19 ribonucleotides, or 20 ribonucleotides in length. Insome embodiments, a sequence-specific endoribonuclease cleavesimmediately 5′ of a recognition sequence. In some embodiments, asequence-specific endoribonuclease cleaves immediately 3′ of arecognition sequence. In some embodiments, a sequence-specificendoribonuclease cleaves within a recognition sequence. In some cases, arecognition sequence is immediately 5′ of a secondary structure. In somecases, a recognition sequence is located 5′ of a secondary structure andwithin 1 nucleotide (nt), 2 nt, 3 nt, 4 nt, 5 nt, or 5 nt to 10 nt ofthe secondary structure. In some cases, a recognition sequence isimmediately 3′ of a secondary structure. In some cases, a recognitionsequence is located 3′ of a secondary structure and within 1 nucleotide(nt), 2 nt, 3 nt, 4 nt, 5 nt, or 5 nt to 10 nt of the secondarystructure.

In some embodiments, the recognition sequence (i.e., RNA recognitionssequence) is derived from an RNA that naturally occurs in the samespecies as that from which the Csy4 polypeptide is derived. In such acase, the RNA recognition sequence is considered a cognate sequence forthe Csy4 polypeptide. For example, FIG. 5 and FIG. 11 depict Csy4polypeptides derived from a variety of species (i.e., naturally occur inthose species). In each case, a cognate RNA recognition sequence (ormultiple different cognate RNA recognition sequences) is listed that isderived from the same species. In some embodiments, the recognitionsequence is derived from an RNA that naturally occurs in a differentspecies as that from which the Csy4 polypeptide is derived (see FIG. 13for examples).

In some embodiments, a substrate polyribonucleotide comprises thestructure X_(x)X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅, where nucleotidesX₁-X₅ base pair with X₁₁-X₁₅ such that X₁ and X₁₅ form the base of astem structure, and such that X₆, X₇, X₈, X₉, and X₁₀ form a loop; thestructure is a regular A-form helical structure.

In some embodiments, the substrate polyribonucleotide comprises anaffinity tag; and a subject method provides for removal of the affinitytag from the substrate polyribonucleotide.

Sequence-Specific Endoribonucleases

Endoribonucleases that bind to and cleave a substrate polyribonucleotidein a sequence-specific manner include enzymatically active polypeptidesthat cleave (hydrolyze) a substrate polyribonucleotide in a metalion-independent fashion.

Structural features of an endoribonuclease that binds to and cleaves asubstrate polyribonucleotide in a sequence-specific and metalion-independent manner can include one or more of the following: 1) ahighly basic alpha helix for sequence non-specific recognition of thephosphate backbone of RNA through the RNA major groove, e.g., R114,R115, R118, R119, or equivalents thereof; 2) R102 and/or Q104, orequivalents thereof, making hydrogen bonding contacts with the majorgroove of the RNA stem; 3) and one or more of His29, Ser148, and Tyr176,or equivalents thereof, involved in catalysis; and 4) F155, or anequivalent thereof.

Endoribonucleases that bind to and cleave a substrate polyribonucleotidein a sequence-specific manner include enzymatically active polypeptidesthat have at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to an amino acid sequence setforth in FIG. 4 (Csy4 amino acid sequences).

Endoribonucleases that bind to and cleave a substrate polyribonucleotidein a sequence-specific manner include enzymatically active polypeptidesthat have at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to an amino acid sequence setforth in FIG. 5 or FIG. 11 (SEQ ID NO: 39, 79, 84, 90, 104, 108, or 110)(Csy4 amino acid sequences).

Endoribonucleases that bind to and cleave a substrate polyribonucleotidein a sequence-specific manner include enzymatically active polypeptidesthat have at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to a Cas6 amino acidsequence.

Endoribonucleases that bind to and cleave a substrate polyribonucleotidein a sequence-specific manner include enzymatically active polypeptidesthat have at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to a CasE amino acidsequence.

Endoribonucleases that bind to and cleave a substrate polyribonucleotidein a sequence-specific manner include enzymatically active polypeptidesthat differ from an amino acid sequence set forth in any one of FIG. 4or 5 by from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20) amino acid substitutions and/orinsertions and/or deletions.

Reaction Conditions

A sequence-specific endoribonuclease can hydrolyze a substratepolyribonucleotide in a sequence-specific manner at a temperature in arange from about 15° C. to about 100° C., e.g., in a range of from about15° C. to about 17° C., from about 17° C. to about 20° C., from about20° C. to about 25° C., from about 25° C. to about 30° C., from about30° C. to about 40° C., from about 40° C. to about 50° C., from about50° C. to about 60° C., from about 60° C. to about 70° C., from about70° C. to about 80° C., from about 80° C. to about 90° C., or from about90° C. to about 100° C.

A sequence-specific endoribonuclease can hydrolyze a substratepolyribonucleotide in a sequence-specific manner in a pH range of fromabout 4.0 to about 8.0, e.g., from about pH 4.0 to about 4.5, from aboutpH 4.5 to about 5.0, from about pH 5.0 to about 5.5, from about pH 5.5to about 6.0, from about pH 6.0 to about 6.5, from about pH 6.5 to about7.0, from about pH 7.0 to about 7.5, from about pH 6.5 to about 7.5,from about pH 7.5 to about 8.0, from about pH 6.5 to about 8.0, or fromabout pH 5.5 to about 7.5.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Direct RNA Detection and Sequencing Using Csy4 Family Proteins

Materials and Methods

Wild-type Csy4, point mutants and selenomethionine (SeMet)-substitutedCsy4 were expressed in Rosetta 2(DE3) cells as either a His₆-maltosebinding protein (MBP) fusion or a His₆ fusion protein and purified byNi-affinity chromatography, followed by proteolytic removal of theHis(MBP) tag, a further Ni-affinity step, and size exclusionchromatography. The pre-crRNAs were transcribed in vitro with T7polymerase and purified on a denaturing gel. The complex was formed byincubating RNA with Csy4 at a 2:1 ratio for 30 minutes at 30° C.followed by size exclusion chromatography. The complex was crystallizedusing the hanging-drop method in 200 mM sodium citrate pH 5.0, 100 mMmagnesium chloride, 20% (w/v) poly(ethylene glycol) (PEG)-4000(wild-type (WT) complex) or 150 mM sodium acetate pH 4.6, 17% PEG4000 or160 mM sodium acetate pH 4.6, 18% PEG4000 (S22C-containing complex). Thestructure of the WT Csy4-RNA complex was determined by themultiwavelength anomalous dispersion (MAD) method usingSeMet-substituted crystals. The structure of the Csy4(S22C)-RNA complexwas determined by molecular replacement.

Gene Annotation, Cloning, Protein Expression and Purification.

Comparative sequence analysis of Csy4 genes across species identified aconserved region 20 codons upstream of the annotated start codon in thePA14 genome. Lee, et al. Genome Biol 7, R90 (2006). The conserved Csy4(PA14_33300) sequence was PCR amplified from Pseudomonas aeruginosaUCBPP-PA14 genomic DNA using Pa14Csy4_fwd: caccatggaccactacctcgacattcgand Pa14Csy4_rev: gaaccagggaacgaaacctcc. The polymerase chain reaction(PCR) product was cloned using the Gateway system into thepENTR/TEV/D-TOPO entry vector (Invitrogen), followed by site-specificrecombination into expression vector pHGWA or pHMGWA. Busso, et al.Analytical Biochemistry 343, 313-321, (2005). Point mutations wereintroduced into Csy4 using the QuikChange Site-Directed Mutagenesis Kit(Stratagene). The Pa14Csy4 expression plasmid was transform into E. coliRosetta 2 (DE3) cells (Novagen) or co-transformed with a pMK vectorexpressing CRISPR RNA synthesized by Geneart (Regensburg, Germany).Rosetta 2 (DE3) cells were grown in Luria Broth (LB) supplemented withampicillin and chloramphenicol. Protein expression was induced with 0.5mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Affymetrix) at a celldensity of ˜0.5OD followed by shaking at 18° C. for 16 hours. Cells werepelleted and resuspended in lysis buffer (15.5 mM disodium hydrogenphosphate, 4.5 mM sodium dihydrogen phosphate, 500 mM sodium chloride,10 mM imidazole, protease inhibitors, 5% glycerol, 0.01% Triton X-100,100μ/ml DNaseI, 1 mM Tris[2-carboxyethyl]phosphine hydrochloride (TCEP),0.5 mM phenylmethylsulfonyl fluoride, pH 7.4) and sonicated on ice fortwo minutes in 10 second bursts. Lysate was clarified by centrifugation(24,000×g, 30 minutes) and incubated with nickel-nitrilotriacetic acid(Ni-NTA) affinity resin in batch (Qiagen). The bound protein was elutedwith high imidazole buffer (15.5 mM disodium hydrogen phosphate, 4.5 mMsodium dihydrogen phosphate, 500 mM sodium chloride, 300 mM imidazole, 1mM TCEP, 5% glycerol, pH 7.4) and dialyzed overnight in dialysis buffer(elution buffer with only 20 mM imidazole) in the presence of tobaccoetch virus (TEV) to cleave the His₆ or His₆MBP tag. The protein wasconcentrated (Amicon) and purified on a nickel affinity column (GE)followed by tandem Sup75 (16/60) columns in gel filtration buffer (100mM HEPES pH 7.5, 500 mM KCl, 5% glycerol, 1 mM TCEP). Sample was thendialyzed against gel filtration buffer containing only 150 mM potassiumchloride. A similar protocol was used for preparation of theselenomethionine (SeMet)-derivitized protein and the only notabledifference was the expression media. Briefly, BL21(DE3) cellstransformed with Csy4(pHGWA) expression vector were grown in M9 minimalmedia supplemented with ampicillin, as previously described. Wiedenheft,et al. Structure 17, 904-912 (2009).

Nuclease Activity Assays.

75 pmol of wild-type or mutant Csy4 were incubated with 5 pmol in vitrotranscribed Pa14 pre-crRNA (prepared as described; Wiedenheft (2009)supra) in 10 μl reactions containing 20 mM HEPES pH 7.5, 100 mMpotassium chloride buffer at 25° C. for five minutes. Reactions werequenched with the addition of 50 ul acid phenol-chloroform (Ambion). 10μl additional reaction buffer were added and samples were centrifuged(16,000×g, 30 minutes) and 16 μl aqueous sample was removed, mixed 1:1with 2× formamide loading buffer, and separated on 15% denaturingpolyacrylamide gel. RNA was visualized with SYBR Gold staining(Invitrogen).

Crystallization.

All crystallization experiments were performed at 18° C. using thehanging drop vapour diffusion method by mixing equal volumes (1 μl+1 μl)of the complex and reservoir solutions. Plate-shaped crystals of thewild-type Csy4-RNA complex were grown in 200 mM sodium citrate pH 5.0,100 mM magnesium chloride, 20% (w/v) poly(ethylene glycol)-4000(PEG4000). These crystals belonged to the space group C2, contained onecopy of the complex in the asymmetric unit and diffracted to 2.3 Åresolution at synchrotron X-ray sources. Using complex reconstitutedwith the Csy4S22C point mutant, two additional crystal forms wereobtained in 150 mM sodium acetate pH 4.6, 17% (w/v) PEG4000 and 160 mMsodium acetate pH 4.6, 18% PEG4000. Initially, hexagonal crystalsappeared within 24 hr. These crystals diffracted to 2.6 Å resolution,belonged to space group P6₁ and contained one copy of the complex in theasymmetric unit. 48 hr later, the same crystallization condition yieldedneedle-shaped crystals that belonged to space group P212121, containedtwo copies of the complex and diffracted up to 1.8 Å resolution. Fordata collection, all crystal forms were cryoprotected by soaking intheir respective mother liquor supplemented with 30% glycerol prior toflash cooling in liquid nitrogen.

Structure Determination.

All diffraction data we collected at 100 K on beamlines 8.2.2 and 8.3.1of the Advanced Light Source (Lawrence Berkeley National Laboratory).Data were processed using XDS. Kabsch, Acta Crystallogr D BiolCrystallogr 66, 125-132 (2010). Experimental phases were determined froma three-wavelength multiwavelength anomalous dispersion (MAD) experiment(peak, inflection and remote data sets) using the monoclinic Csy4-RNAcrystals containing selenomethionine-substituted wild-type Csy4. Twoselenium sites were located using the Hybrid Substructure Search (HySS)module of the Phenix package. Grosse-Kunstleve, and Adams. ActaCrystallogr D Biol Crystallogr 59, 1966-1973 (2003). Substructurerefinement, phasing and density modification were performed usingAutoSHARP. Vonrhein, et al. Methods Mol Biol 364, 215-230 (2007). Theresulting electron density map exhibited clear layers of densityattributable to protein and RNA alternating along the c-axis, with theRNA layer made up of two coaxially-stacked RNA helices engaged in a“kissing loop” interaction. An initial atomic model for the Csy4 proteinwas obtained by automatic building using the Phenix AutoBuild module.Terwilliger, et al. Acta Crystallogr D Biol Crystallogr 64, 61-69,(2008). The complex model was completed by iterative cycles of manualbuilding in COOT (Emsley, and Cowtan, Acta Crystallogr D BiolCrystallogr 60, 2126-2132 (2004)) and refinement using Phenix.refine³⁶(Adams, et al. Acta Crystallogr D Biol Crystallogr 66, 213-221 (2010))against a native 2.33 Å resolution dataset, yielding a final model witha crystallographic R_(work) factor of 21.4% and a R_(free) factor of26.4% (Table 1).

TABLE 1 Data collection, phasing and refinement statistics Native NativeNative WT S22C S22C SeMet WT Data collection Space group C2 P2₁2₁2₁ P6₁C2 Cell dimensions a, b, c (Å) 62.37, 72.77, 40.1, 78.9, 39.25, 39.25,62.33, 47.23, 86.82 145.9 297.37 87.26 α, β, γ (°) 90.0, 108.2, 90.0,90.0, 90.0, 90.0, 90.0, 108.3, 90.0 90.0 120.0 90.0 Peak InflectionRemote Wavelength (Å) 1.11159 0.99992 1.11588 0.97949 0.97971 0.97204Resolution (Å)* 19.68-2.33 69.4-1.80 22.38-2.60 82.86-2.80 82.86-2.8082.86-2.80 (2.50-2.33) (1.90-1.80)  (2.70-2.60)  (2.90-2.80) (2.90-2.80)  (2.90-2.80) R_(sym) (%)*  5.8 (44.6)  7.0 (52.8)  3.3(31.1)  9.4 (38.3)  8.9 (38.5)  9.0 (38.1) I/σ/* 18.9 (3.35) 31.1 (3.1)29.8 (3.8)  17.0 (4.4)  14.5 (3.7)  14.5 (3.6)  Completeness (%)* 96.6(98.3)  98.7 (91.0) 99.4 (98.5) 99.6 (96.7) 99.5 (99.3) 99.3 (96.4)Redundancy* 4.4 (4.4) 19.8 (6.5) 6.1 (5.4) 5.7 (5.3) 3.8 (3.7) 3.8 (3.7)Refinement Resolution (Å) 19.70-2.33 69.4-1.80 19.60-2.60 No.reflections 9974 43284 7798 R_(work)/R_(free) 0.214/0.265 0.187/0.2200.255/0.279 No. atoms Protein 1273 2975 1364 RNA 313 642 321Water/ligands 41 386 5 B-factors Protein 47.7 29.1 101.5 RNA 109.3 35.3103.0 Water/ligands 44.9 33.5 74.5 R.m.s. deviations Bond lengths (Å)0.007 0.011 0.002 Bond angles (°) 1.0 1.5 0.7 *Values in parenthesesdenote highest resolution shell

The model includes RNA nucleotides C1-G15 and the phosphate group ofnucleotide C16 and protein residues 1-104, 109-120 and 139-187. Owing tothe layered arrangement of protein and RNA in the crystal lattice andthe lack of lateral crystal contacts within the RNA layer, the RNAexhibits significant disorder, as evidenced by markedly elevatedtemperature factors (>100 Å²) and the absence of interpretable densityfor the nucleotide base of U9. The disorder is also evident in proteinresidues 109-120, corresponding to the arginine-rich helix inserted inthe major groove of the RNA, for which only the polypeptide backbonecould be built (except for residues Arg 115 and Arg 118).

The structures of the Csy4(S22C)-RNA complex in the hexagonal andorthorhombic crystal forms were determined by molecular replacement inPhaser (McCoy, et al. J Appl Crystallogr 40, 658-674 (2007)), using theCsy4 protein (lacking the arginine-rich helix) and RNA models from themonoclinic crystal form as separate search ensembles. In both crystalforms, electron density for the arginine-rich helix and the linkerregion comprising Csy4 residues 105-108 was immediately noticeable in2F_(o)-F_(c) maps obtained from the molecular replacement solutions. Thestructure of the Csy4(S22C)-RNA complex in the hexagonal form wasrefined to an R_(work) factor of 25.5% and R_(free) of 27.9 at 2.6 Åresolution. The final model includes Csy4 residues 1-120 and 139-187 andRNA nucleotides C1-G15 plus the phosphate group of nucleotide C16. Theorthorhombic crystal form of the Csy4(S22C)-RNA complex has been solvedat 1.8 Å resolution and refined to an R_(work) factor of 18.7% andR_(free) of 22.0%, with excellent stereochemistry. Of the two complexesin the asymmetric unit, complex 1 (chains A and C) contains Csy4residues 1-187 and RNA nucleotides C1-G15 plus the phosphate group ofnucleotide C16, while the less ordered complex 2 (chains B and D)comprises Csy4 residues 1-187 with the exception of residues 13-15 and135-138, which show no ordered electron density, and RNA nucleotidesC1-G15 and the phosphate group of nucleotide C16. The two copies of Csy4superpose with an rmsd of 1.15 Å over 179 Cα atoms, the greatestdifferences coming from the slightly different positions of thearginine-rich helix. The two RNA molecules in the asymmetric unitsuperpose with an rmsd of 1.49 Å, the largest deviation being due to thebulged-out nucleotide U9, which assumes different conformations in thetwo RNAs. Our discussion and illustrations throughout the manuscript arebased on complex 1 of the orthorhombic crystal form. All structuralillustrations were generated using Pymol (http://www(dot)pymol(dot)org).

Results

CRISPR-mediated immunity is thought to occur in approximately 90% ofarchaeal and 40% of bacterial genomes based on the presence of CRISPRloci in sequenced genomes. Horvath and Barrangou, Science 327, 167-170(2010); Jansen, et al. Molecular Microbiology 43, 1565-1575 (2002);Sorek, et al. Nat Rev Microbiol 6, 181-186 (2008); Marraffini, andSontheimer, Nat Rev Genet 11, 181-190 (2010). CRISPR-associated (Cas)proteins belonging to the eight known CRISPR/Cas subtypes are highlydivergent at the primary sequence level, obscuring identification offunctional homologues. Haft, et al. PLoS Comput Biol 1, e60 (2005);Makarova, et al. Biology Direct 1, 1-26 (2006). Pseudomonas aeruginosaUCBPP-PA14 (hereafter Pa14), a Gram-negative opportunistic pathogenharboring a CRISPR/Cas system of the Yersinia subtype, contains six Casgenes flanked by two CRISPR elements (FIG. 1A). Although Cas1 is founduniversally among CRISPR-containing organisms, and Cas3 is evident inmost subtypes, Csy1-4 are unique to the Yersinia subtype. Both CRISPRelements comprise a characteristic arrangement of 28-nucleotide repeatsidentical within both CRISPRs (save for one nucleotide) interspersedwith ˜32-nucleotide unique spacers, some of which match sequences foundin bacteriophage or plasmids. Grissa, et al. BMC Bioinformatics 8, 172(2007). In many organisms it has been shown that CRISPR loci aretranscribed as long single units and are post-transcriptionallyprocessed to yield crRNAs that each contain one unique sequence flankedby sequences derived from the repeat element. Brouns et al. Science 321,960-964 (2008); Carte, et al. Genes and Development 22, 3489-3496(2008); Tang, et al. Proc. Natl. Acad. Sci. USA 99, 7536-7541 (2002);Lillestol, et al. Archaea 2, 59-72 (2006); Lillestol, et al. MolMicrobiol 72, 259-272 (2009); Tang, et al. Molecular Microbiology 55,469-481 (2005).

To identify the protein(s) responsible for producing crRNAs from longCRISPR transcripts (pre-crRNAs) in the Yersinia subtype, each of the sixCas proteins from Pa14 was recombinantly expressed, and therecombinantly expressed proteins were tested for endoribonucleolyticfunction using an in vitro transcribed pre-crRNA. Based onsequence-specific pre-crRNA processing activity, it was found that Csy4is the endoribonuclease responsible for crRNA biogenesis. As observedfor crRNA processing within two other CRISPR/Cas subtypes (Brouns et al.(2008) supra; Carte et al. (2008) supra), CRISPR transcript cleavage isa rapid, metal ion-independent reaction. Csy4 cleaves pre-crRNA withinthe repeat element at the base of a predicted stem-loop structure,generating ˜60 nucleotide crRNAs consisting of a 32-nucleotide unique(phage-derived) sequence flanked on the 5′ and 3′ ends by eight and 20nucleotides, respectively, of repeat sequence (FIG. 1A).

For Csy4 to be effective, it was hypothesized that its RNA recognitionmechanism must be highly specific in order to target only CRISPR-derivedtranscripts and not other cellular RNAs containing hairpins and/orrelated sequences. To test this, Csy4 was expressed in E. coli alone orco-expressed with a Pa14 CRISPR RNA. In spite of a high isoelectricpoint (PI=10.2), Csy4 does not associate with cellular nucleic acids;however, when co-expressed with a Pa14 CRISPR, the protein is associatedwith a crRNA (FIG. 1B,C). These observations underscored the specificityof Csy4 recognition, leading us to explore the protein/RNA interactionsrequired for Csy4 substrate recognition and cleavage. Csy4 binding andactivity assays were performed in vitro using RNA oligonucleotidescorresponding to different regions of the 28-nucleotide Pa14 CRISPRrepeat sequence. Using this approach, a minimal RNA fragment recognizedby Csy4 consisting of the repeat-derived stem-loop and one downstreamnucleotide was identified. Cleavage assays utilizing this minimal RNA asa substrate showed that Csy4 activity requires a 2′OH on the riboseimmediately upstream of the cleavage site. A 2′-deoxyribose at thisposition completely abrogates cleavage, but does not disrupt Csy4binding.

In order to obtain structural insights into crRNA recognition andcleavage, Csy4 was co-crystallized in complex with a minimal RNAsubstrate. To generate a stable complex for structural analysis, Csy4was bound to the non-cleavable 16-nucleotide minimal RNA substratedescribed above in which the nucleotide preceding the cleavage site is a2′-deoxynucleotide. Crystals of the complex were obtained in threeunique space groups, each exhibiting different crystal packing; onecontained wild-type Csy4 and two contained a Csy4 point mutant. Thecrystal structure of the Csy4-RNA complex was solved to a resolution of1.8 Å (FIG. 2A, Table 1), revealing an unanticipated mechanism by whichCRISPR RNA is recognized and processed for use by the CRISPR-mediatedsilencing machinery. Csy4 makes sequence-specific contacts in the majorgroove of the stem-loop of the CRISPR repeat sequence and additionalsequence non-specific contacts with the phosphate backbone of the RNAstem. The majority of characterized protein/RNA interactions aremediated via the minor groove of an RNA helix; the recognition of theRNA major groove by Csy4 is a highly unusual mechanism of protein/RNAinteraction.

At the primary sequence level, Csy4 is highly dissimilar from the otherknown endoribonucleases involved in crRNA biogenesis (CasE from Thermusthermophiles (Ebihara, et al. Protein Sci 15, 1494-1499 (2006)) and Cas6from Pyrococcus furiosus Carte et al. (2008) supra), sharing only ˜10%identity. The crystal structures of both CasE and Cas6 indicate thatthese proteins adopt tandem ferrodoxin-like folds. Notably, Csy4 sharesthis fold with these enzymes; in the Csy4-RNA complex, the N-terminaldomain (residues 1-94) of Csy4 indeed adopts a ferredoxin-like fold.However, although the C-terminal domain (residues 95-187) shares thesame secondary structure connectivity as a ferredoxin-like fold, but itsconformation is markedly different. Strikingly, an arginine-rich helix(residues 108-120) from the putative C-terminal ferredoxin domaininserts into the major groove of the hairpin RNA. Structuralsuperpositions using the DALI server (Holm, and Sander, J Mol Biol 233,123-138 (1993)) indicate that Csy4 in its RNA-binding conformationsuperposes with CasE and Cas6 with root-mean-square deviation (rmsd) of3.8 Å (over 111 Ca atoms) and 3.9 Å (over 104 Ca atoms), respectively.Csy4, CasE and Cas6 could be descendants of a single ancestralendoribonuclease that has diverged markedly at the sequence level as itco-evolved with the repeat sequence of the CRISPR locus, whilemaintaining a similar protein fold.

The crRNA substrate forms a hairpin structure, as predicted for thissubclass of crRNA repeats (Kunin, et al. Genome Biol 8, R61 (2007)),with nucleotides 1-5 and 11-15 base pairing to produce a regular A-formhelical stem. The GUAUA pentaloop contains a sheared G6-A10 base pairand a bulged-out nucleotide U9, its structure reminiscent of GNR(N)Apentaloops found in the yeast U6 small nuclear RNA intramolecularstem-loop (Huppler, et al. Nat Struct Biol 9, 431-435 (2002)) and inbacteriophage lamda BoxB RNA (Legault, et al. Cell 93, 289-299 (1998)).In the Csy4-RNA complex, the RNA stem-loop straddles the β-hairpinformed by strands β7-β8 of Csy4, with the C1-G15 base-pair directlystacking onto the aromatic side chain of Phe155 (FIG. 2B). This anchorsthe RNA stem and orients it at the proper angle to permitsequence-specific interactions in the major groove.

Two residues in a linker segment connecting the body of Csy4 to thearginine-rich helix, Arg102 and Gln104, make hydrogen bonding contactsin the major groove of the RNA stem, sequence-specifically recognizingG15 and A14, respectively (FIG. 2B). The Csy4-crRNA interaction isfurther stabilized by the insertion of the arginine-rich helix into themajor groove of the RNA hairpin in the proximity of the bulged-outnucleotide U9 (FIG. 2C). The side chains of Arg 114, Arg 115, Arg 118and Arg119 contact the phosphate groups of nucleotides 2-6.Additionally, the sidechain of Arg115 engages the base of G6 as the onlysequence-specific interaction between the arginine-rich helix and theRNA hairpin. Interestingly, this interaction is highly reminiscent ofhow certain viral proteins interact with the major groove of dsRNAmolecules, for example the Tat/Tar interaction in human immunodeficiencyvirus (HIV)²³ and the lambda-N/boxB complex in lambdoid phages (Cai, etal. Nature Structural Biology 5, 203-212 (1998)). In both cases, ahighly basic α-helix is employed for sequence non-specific recognitionwith the phosphate backbone of RNA through the RNA major groove.

Csy4 recognizes the hairpin element of the CRISPR repeat sequence andcleaves immediately downstream of it. The structure described in thisExample contains a substrate-mimic RNA, which is not competent forcleavage. In the active site, density was observed only for thephosphate group 3′ of the penultimate nucleotide, but no density for theterminal sugar or base, presumably due to the flexibility of thisnucleotide (FIG. 3A). The scissile phosphate binds in a pocket locatedbetween the β-turn of the β7-β8 hairpin on one side and helix al and aglycine-rich loop, previously identified in Cas6 and CasE, on the other.Three residues proximal to that phosphate group are likely toparticipate in catalysis, His29, Ser148 and Tyr176. These residues areinvariant among 12 Csy4 sequences that were identified using a BLASTsearch (Altschul, et al. Nucleic Acids Research 25, 3389-3402 (1997))coupled with manual verification of a nearby CRISPR locus (Grissa, etal. BMC Bioinformatics 8, 172 (2007)) (FIG. 4).

The structure suggests that several residues in Csy4 are important formediating substrate recognition/binding and catalysis. Point mutants ofeach of these residues were generated; their cleavage activity wastested biochemically (FIG. 3B). Mutation of putative catalytic siteresidues His29 or Ser148 abolishes cleavage activity. However, mutationof Tyr176 to phenylalanine does not disrupt activity, indicating thatTyr176 may play a crucial role in orienting His29, though it does notdirectly participate in catalysis. Mutation of Arg102 to alanineabolishes accumulation of crRNAs, whereas mutation of Gln104 to alaninedoes not significantly disrupt activity, suggesting that Arg102, whichrecognizes the terminal base pair, is important for properly orientingthe RNA substrate, but that Gln104 is not required for in vitroactivity. Phe155 appears to play a large role in appropriately orientingthe RNA substrate, as an alanine mutation at this residue severelyimpairs crRNA biogenesis.

The identification of a serine involved in mediating RNA cleavage isunexpected. Although mutation of His29 to alanine results in acatalytically inactive Csy4, mutation to lysine partially restoresactivity, strongly suggesting that His29 acts as a proton donor, not toinitiate cleavage via a nucleophilic attack.

CRISPRs are the genetic memory of a nucleic acid-based immune systemthat relies on small CRISPR-derived RNAs for guiding the immune systemto cognate sequences associated with invading genetic elements.Phylogenetic analysis of CRISPR repeat sequences has identified distinctCRISPR categories (Kunin, et al. Genome Biol 8, R61 (2007)) thatcorrelate with a particular set of Cas genes. The co-variation of Casgenes with specific CRISPR repeat sequence types suggests that CRISPRrepeats have co-evolved with the Cas genes that are responsible forCRISPR adaptation, the generation of crRNAs and the silencing ofinvading genetic elements. The structure described here details anunusual recognition mechanism that discriminates crRNA substrates basedon both sequence- and structure-specificity, providing great insightinto the ability of Csy4 and its homologues to readily distinguishsubstrate RNA from among all cellular RNAs.

FIGS. 1A-C. Pa14Csy4 specifically recognizes only its pre-crRNAsubstrate. a, Schematic of CRISPR/Cas locus in Pa14. The six Cas genesare flanked on both sides by CRISPR loci. Enlarged is a schematicshowing the predicted stem-loop in the 28-nucleotide direct repeat(black lettering) separated by 32-nucleotide spacer sequences (blue).The red arrows note the bond cleaved by Csy4. b,c Comparison of protein(b) and RNA content (c) after Pa14Csy4 expression in E. coli with (+)and without (−) a plasmid containing a Pa14 CRISPR locus. Purified Csy4from both preparations was split into two pools. Half were resolved onSDS-PAGE and visualized with Coomassie blue staining; half were acidphenol-chloroform extracted, resolved on UREA-PAGE, and visualized withSYBR Gold (Invitrogen).

FIGS. 2A-C. The crystal structure of Csy4 bound to RNA substrate. a,Front and back views of the complex. Csy4 is colored in blue and the RNAbackbone is colored in orange. b, Detailed interactions between residuesR102 and Q104 and nucleotides A14 and G15. Hydrogen bonding is depictedwith dashed lines. c, Detailed interactions between an arginine-richalpha helix and the RNA backbone and G6.

FIGS. 3A and 3B. Putative active site. a, Detailed view of the catalyticcenter. b, Cleavage activity of Csy4. Wild-type (WT) Csy4 and a seriesof single point mutants were incubated with in vitro transcribedpre-crRNA for 5 minutes at 25 C. Products were acid phenol-chloroformextracted and resolved on UREA-PAGE and visualized by SYBR Goldstaining.

Example 2 Direct RNA Sequencing

An RNA can be sequenced at the single-molecule level using FörsterResonant Energy Transfer (FRET). The RNA to be sequenced will beattached to a solid surface through its 3′ ribose. The RNA should bespaced far enough from neighboring RNA molecules on the surface to allowdetection at the single-molecule level. The spacing is dictated bydiffraction-limited methods, dependent on the wavelength of emittedlight. Alternatively, the RNA spacing can be closer than the diffractionlimit, if super-resolution imaging methods are used. In the firstsequence detection step, a Csy4 family protein of known nucleic acidbinding specificity is added to the RNA to be sequenced, along with apool of detection oligonucleotides. The Csy4 protein will only bind tothe RNA to be sequenced if one of the detection oligonucleotides canform a 4 base pair double helix with the RNA to be sequenced. Inaddition, the detection nucleotide must base pair with an additional 3nucleotides 3′ of the 4 base pair recognition sequence in the RNA to besequenced, in order for the Csy4 protein to bind stably. The detectionoligonucleotides will contain an extension of 3 nucleotides 3′ of the4-nucleotide recognition sequence. In the pool of detectionoligonucleotides, the 3-nucleotide extension will have a defined 5′nucleotide followed by two random nucleotide positions; or a randomnucleotide at the 5′ position followed by a defined nucleotide and arandom nucleotide; or 2 random nucleotides at the 5′ end, followed by adefined nucleotide. In any of these pools, the defined nucleotide isknown based on an attached fluorescent molecule, the emission orexcitation spectrum of which is defined by the nucleotide. The Csy4protein will be attached to a quantum dot whose excitation spectrumoverlaps with the emission spectrum of the fluorescent molecule attachedto the detection oligonucleotide. After binding of detectionoligonucleotides and Csy4, excess reagents will be washed away. Apositive binding event is detected only if the detection nucleotideforms a 7-nucleotide double helix with the RNA to be sequenced. Ifbinding occurs, the resulting ternary complex of RNA to be sequenced,detection oligonucleotide, and Csy4 protein can be detected by FRET fromthe fluorescent molecule attached to the detection oligonucleotide tothe quantum dot attached to the Csy4 protein. After each cycle ofbinding, the Csy4 protein and detection oligonucleotides will be removedfrom the sample using chemical and/or heat denaturation and washing. Insubsequent sequencing steps, other Csy4 proteins of different sequencespecificity and their corresponding detection oligonucleotides will beincubated with the RNA to be sequenced, in a similar manner. Othervariations of the 3-nucleotide extension on the detectionoligonucleotide can be envisioned, such as extensions of differentlengths, at either the 5′ end or 3′ end of the detectionoligonucleotide. The detection oligonucleotide could be RNA, DNA, or anychemically modified version of these polymers, such as PNAs or LNAs.

Example 3 Inducible Sequence-Specific Endoribonuclease

Via biochemical and structural techniques, point mutants of Csy4 thatlack cleavage activity, while retaining substrate binding activity, havebeen generated. An example is the above-described Csy4(H29A) mutant. Theotherwise catalytically inactive Csy4(H29A) mutant can be reactivated inthe presence of exogenous imidazole. Addition of between 150 mM and 300mM imidazole to the reaction buffer is sufficient to stimulate near-wildtype cleavage activity. The results are shown in FIG. 8. FIG. 8 shows acleavage activity assay depicting the imidazole rescue. Csy4H29A is acatalytically inactive mutant of Csy4 that retains the ability to bindits substrate with a kd of <1 nM.

Reaction details for FIG. 8: Each 10 μl reaction contains 5 pmol of thein vitro transcribed pre-crRNA substrate, 100 pmol of Csy4 (WT or H29A,as indicated in FIG. 8), 20 mM HEPES pH 7.5, 100 mM KCl, and 150-300 mMimidazole, as indicated. Reactions were carried out for 30 minutes at25° C.) Products were acid phenol-chloroform extracted, separated on a15% denaturing gel, and visualized with SYBR Gold. Biochemicalcharacterization of Csy4(H29A) shows that it binds to its RNA substratewith <1 nM affinity.

FIG. 11 depicts additional examples of sequence-specific Csy4endoribonucleases and mutant, inactive versions of theseendoribonucleases that can be reactivated in the presence of imidazole.The residue to be mutated (His to Ala) is inferred from the conservedhistidine (asterisk) revealed by the alignment depicted in FIG. 4. As anon-limiting example, FIG. 11D depicts that a corresponding H29A (His toAla) mutation according to a sequence alignment such as that depicted inFIG. 4 (which can be readily determined by one of ordinary skill in theart for any Csy4 protein) can be H34A when generating an inactiveversion (that can be reactivated in the presence of imidazole) of Csy4(SEQ ID NO: 109, Ala-34) from an active version of Csy4 (SEQ ID NO: 108,His-34) from, for example, Acinetobacter sp. ADP1.

The results are shown in FIG. 12. FIG. 12 shows results from five Csy4enzymes that were mutated to generate active site His to Ala mutations:(A) Ec89, Escherichia coli UTI89; (B) Dn, Dichelobacter nodosus; (C) AA,Acinetobacter sp. ADP1; (D) SspW3, Shewanella sp. W3-18-1; and (E) Ab,Acinetobacter baumannii. Reaction details for FIG. 12: 75 pmol ofwild-type or mutant proteins were incubated with 50 pmol of one or twoof the RNA substrates (see FIG. 11) that co-occur with these proteinsequences in the absence or presence of 300 mM imidazole in 20 μlreactions. Reactions were carried out at room temperature for 30 minutes(B,D) or 3 hours (A,C,E). Samples were phenol:chloroform extracted,separated on 20% denaturing gels, and stained with SYBR-Gold.

Csy4(H29A) is useful for both in vivo and in vitro applications forwhich there is no current alternative approach.

Csy4(H29A) or a corresponding variant as described above (also referredto herein as “inducible” Csy4), is useful for purifying a particularRNA/protein complex (RNP) from a complex mixture of RNAs and RNPs(RNA/protein complexes). For example, researchers may be interested inunderstanding which proteins bind to a particular RNA transcript. Usingthis system, the researchers could engineer an expression construct fortheir RNA of choice that would include a 5′ tag consisting of thestem-loop Csy4 target sequence. The researchers would then transfectthis expression construct into their cell type of choice, leading to thegeneration of many RNAs and RNPs. Cells would then be lysed and thelysate would be applied to a column that contains inducible Csy4immobilized on agarose beads. RNAs or RNPs that have the Csy4 targetsequence will bind. A subsequent wash step will remove non-specificallybound RNAs. A wash with imidazole (˜300 mM) will activate inducibleCsy4, which will cleave the target sequence and release the boundRNA/RNP. This method is illustrated schematically in FIG. 9.

A similar method could be useful for assembling RNPs in vitro. Forexample, an RNA of choice could be transcribed in vitro using aconstruct similar to the expression plasmid designed for the aboveexperiment. (The construct must introduce the Csy4 stem-loop targetsequence at the 5′ end of the transcribed RNA.) This in vitrotranscribed product could then be incubated with proteins known orsuspected to bind the particular transcript. The inducibleCsy4-containing column could be used to purify the in vitro formed RNPsaway from free protein.

Example 4

The mechanism for specific substrate recognition by the endoribonucleaseCasE, an essential component of the CRISPR immune system found in themajority of bacteria and archaea (van der Oost et al., Trends BiochemSci. 34, 401-7 (2009)) has been determined. Using structural andbiochemical methods, the minimal RNA sequence required for optimalsubstrate cleavage, a 20 nucleotide sequence (5-24 of CRISPR repeatsequence) that includes a seven base-pair stem-loop followed by twounpaired nucleotides, was identified. The structure of this RNA bound toCasE from Thermus thermophilus was solved at 2.0 Å resolution usingX-ray crystallography. This structure reveals numerous sequence specificcontacts between the protein and RNA, including several interactions inthe major groove of the RNA. The terminal base-pair in the stem-loop indisrupted, with A22 flipped out of the helix and base-stacked with U23.This conformation is partially stabilized by interactions with S34 andE38, which also confer sequence specificity for substrate recognition.Further stabilization of the A22 and U23 conformation is achieved bypositioning of the terminal nucleotide, G24, which flips back intoregister with the stem-loop, but resides well below the helix in abinding pocket made up of residues D18, E24, and K31, with R27contacting the backbone between U23 and G24.

The positioning of A22 elongates the backbone of the RNA at the scissilephosphate, splaying it between two active site residues, Y23 and H26.Based on this observation, and the apparent stabilization of this RNAconformation by G24 binding, it was hypothesized that G24 may berequired for positioning the RNA in a catalytic conformation. Consistentwith this hypothesis, deletion or mutation of G24 significantly reducescleavage activity, as does mutations of protein residues involved in G24binding. To confirm the role of G24 in inducing the catalytic RNAconformation, the structure of CasE bound to a 19 nucleotide RNA thatlacked the terminal G24 residue was determined. This complexcrystallized in two different forms, which revealed two different RNAconformations at the active site of the protein. In one crystal form(P2₁), the 2.5 Å structure contained 8 molecules in the asymmetric unit.All 8 molecules revealed that A22 base stacks with G21, maintainingA-form geometry with the rest of the stem-loop. In addition to thechanges in the RNA structure, the protein structure also differs fromthe catalytic conformation observed in the 2.0 Å structure. In thatstructure, a loop containing R158 and K160 is juxtaposed with the activesite, suggesting that these residues may play a role in catalysis or instabilization of a transition-state intermediate. In the 2.5 Åstructure, this loop is distal from the active site and partiallydisordered, suggesting that the positioning of the loop is flexible.Interestingly, this loop is also disordered in the apo structure of CasE(Ebihara et al., Protein Sci. 15, 1494-9 (2006)), suggesting that thecorrect RNA conformation is required for stabilization of this loop.

The second crystal form (P2₁2₁2₁) obtained for the CasE/19-nucleotideRNA complex was used to determine a 1.5 Å structure, which revealed theRNA bound in the catalytic conformation with A22 and U23 flipped out ofthe helix. However, the loop containing R158 and K160 remains disorderedin this structure, suggesting that G24 binding may also be required forstabilization of this protein structure. The observation of twodifferent RNA conformations for the same complex suggests that the RNAmay sample several structural states, and that it may require G24 tolock it into the catalytically competent conformation.

Example 5

FIG. 13 illustrates that Csy4 endoribonuclease from a given species cancleave RNA substrates from various other species with rate constantsthat are either greater or less than that achieved when cleaving acognate (from the same species) RNA substrate. (A) In vitro cleavagereactions were carried out with each of six Csy4 variants paired witheither its own cognate RNA(s) or one of the variant RNA sequences. AllRNA substrates used in this experiment were the long form. For proteinand RNA sequences, see FIGS. 11A-G) (Pa14, Pseudomonas aeruginosa; Ec89,Escherichia coli UTI89; Dn, Dichelobacter nodosus; AA, Acinetobacter sp.ADP1; Ab, Acinetobacter baumannii; MM, Marinomonas sp. MWYL1; and SspW3,Shewanella sp. W3-18-1). Values shown are the reaction rate constant(per minute). Reactions contained 50 nM Csy4, 0.1-1 nM RNA, 20 mM HEPES,100 mM KCl, and 1 mM DTT at pH_(RT) 7.5. (B) Rates are normalized foreach Csy4 homolog tested in FIG. 11, such that the largest rate constantin each row is defined as 100%.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An in vitro method of regulating production of atarget RNA from a precursor RNA in a eukaryotic cell, the methodcomprising: introducing into said eukaryotic cell a recombinantexpression vector comprising a nucleotide sequence encoding anenzymatically active sequence-specific Csy4 endoribonuclease, whereinthe Csy4 endoribonuclease comprises an amino acid sequence having atleast 95% amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:8, wherein the target RNA comprises aGUUCACUGCCGUAUAGGCAG (SEQ ID NO:103) cleavage site in the 3′untranslated region of a precursor RNA comprising the target RNA, andwherein said Csy4 endoribonuclease is produced in the cell and cleavessaid target RNA from the precursor RNA at the cleavage site, therebyregulating production of the target RNA.
 2. The method of claim 1,wherein the target RNA species is a regulatory RNA.
 3. The method ofclaim 1, wherein cleavage of said target RNA from a precursor RNAinactivates the precursor RNA.
 4. The method of claim 1, wherein saidnucleotide sequence encoding an enzymatically active sequence-specificCsy4 endoribonuclease is operably linked to a promoter.
 5. The method ofclaim 4, wherein said method comprises contacting the promoter with anagent that activates said promoter.
 6. The method of claim 4, whereinsaid cleaving of said target RNA from said precursor RNA inactivatessaid target RNA.
 7. The method of claim 4, wherein said cleaving of saidtarget RNA from said precursor RNA reduces expression levels of saidtarget RNA.
 8. The method of claim 4, wherein said precursor RNAcomprises a poly-A tail.
 9. The method of claim 4, wherein the Csy4endoribonuclease comprises an amino acid sequence having at least 98%amino acid sequence identity to the amino acid sequence set forth in SEQID NO:
 8. 10. The method of claim 4, wherein the Csy4 endoribonucleasecomprises a moiety that provides a detectable signal.
 11. The method ofclaim 10, wherein the moiety is a fluorophore, a quantum dot, an enzymeother than the endoribonuclease, or a nanoparticle.
 12. The method ofclaim 4, wherein the Csy4 endoribonuclease binds an RNA substratecomprising the nucleotide sequence 5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′(SEQ ID NO: 1).
 13. The method of claim 4, wherein said eukaryotic cellis a primary cell.
 14. The method of claim 4, wherein said eukaryoticcell is a mammalian cell.